Yersinia spp. polypeptides and methods of use

ABSTRACT

The present invention provides isolated polypeptides isolatable from a  Yersinia  spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

CONTINUING APPLICATION DATA

This application is a continuation patent application of U.S. patentapplication Ser. No. 14/933,899 filed on Nov. 5, 2015; which is acontinuation patent application of U.S. patent application Ser. No.14/793,494, filed on Jul. 7, 2015, now U.S. Pat. No. 9,221,899; which isa continuation patent application of U.S. patent application Ser. No.14/048,649, filed on Oct. 8, 2013, now U.S. Pat. No. 9,085,613; which isa continuation patent application of U.S. patent application Ser. No.11/336,706, filed on Jan. 20, 2006, now U.S. Pat. No. 8,563,004; whichclaims the benefit of U.S. Provisional Application No. 60/646,106, filedJan. 21, 2005, each of which is incorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext filed entitled “29300410108_SequenceListing_ST25.txt” having a sizeof 195 kilobytes and created on Apr. 27, 2016. Due to the electronicfiling of the Sequence Listing, the electronically submitted SequenceListing serves as both the paper copy required by 37 CFR §1.821(c) andthe CRF required by §1.821(e). The information contained in the SequenceListing is incorporated by reference herein.

BACKGROUND

There are three Yersinia species that are pathogenic to humans: Y.pestis, Y. pseudotuberculosis, and Y. enterocolitica. Y. pestis is thecausative agent of plague, while Y. pseudotuberculosis and specificpathogenic serovars of Y. enterocolitica cause gastrointestinalillnesses. Other species of Yersinia, including Y. rohdei, Y. aldovae,Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. kristensenii, and Y.moolaretti, are considered enterocolitica-like opportunist pathogenswith the ability to cause diarrheal illness in susceptible individuals(Agbonlahor, J Clin Microbiol, 23, 891-6, (1986), Cafferkey, et al., JHosp Infect, 24, 109-15, (1993), Loftus, et al., Dig Dis Sci, 47,2805-10, (2002)). The Yersinia can also infect other animal speciescausing a range of illnesses. Most wild and domestic species of mammalsare prone to infections with the enteropathogens Y. enterocolitica andY. pseduotuberculosis, although most of these infections are subclinicaland such animals usually serve only as asymptomatic carriers of thepathogens for transmission to humans (Fantasia, et al., J ClinMicrobiol, 22, 314-5, (1985), Fantasia, et al., Vet Rec, 132, 532-4,(1993), Fukushima. et al., J Clin Microbiol, 18, 981-2, (1983),Kageyama, et al., J Med Primatol, 31, 129-35, (2002), Kato, et al., ApplEnviron Microbiol, 49, 198-200, (1985), Poelma, et al., Acta Zool PatholAntverp, 3-9, (1977), Shayegani, et al., Appl Environ Microbiol, 52,420-4, (1986), Yanagawa, et al., Microbiol Immunol, 22, 643-6, (1978).).However, there are reports that the enteropathogenic Yersinia have beenassociated with diarrheal illness and general malaise in domesticanimals such as sheep, cattle, goats, pigs, dogs, birds and farmed deer(Jerrett, I. V., et al., Aust Vet J, 67, 212-4, (1990). Slee, K. J., etal., Aust Vet J, 65, 271-5, (1988), Slee, K. J. and C. Button, Aust VetJ, 67, 396-8, (1990), Slee, K. J. and C. Button, Aust Vet J, 67, 320-2,(1990), Zheng, X. B., J Appl Bacteriol, 62, 521-5, (1987)). Y. pestiscan cause disease in a variety of rodent species as well as nonhumanprimates (Davis, K. J., et al., Arch Pathol Lab Med, 120, 156-63,(1996), Meyer, K. F., et al., J Infect Dis, 129, Suppl:S85-12, (1974).Y. pestis is also associated with potentially severe infections indomestic cats (Gasper, P. W., et al., J Med Entomol, 30, 20-6, (1993))and a few cases of Y. pestis infection have been reported in dogs(Orloski, K. A. and M. Eidson, J Am Vet Med Assoc, 207, 316-8, (1995)).In addition, Yersinia ruckeri is a pathogen of fish, causing redmouthdisease in salmonids (Furones, M. D., et al., Ann. Rev. Fish Dis., 3,105-125, (1993)).

Plague is undoubtedly one of the most devastating acute infectiousdisease in the recorded history of man, estimated to have killed 100 to200 million people worldwide (Perry, R. D. and J. D. Fetherston, ClinMicrobiol Rev, 10, 35-66, (1997)). In recent years plague outbreaks havebeen relatively uncommon in the U.S. and other industrialized countries,although endemic foci exist in all continents except Australia.Worldwide surveys indicated 2000 to 5000 annual cases of plague reportedin the last several years, although epidemiologists suspect that manyhuman cases of plague are unreported. Y. pseudotuberculosis outbreaksare fairly rare, and have occurred primarily in Finland, Japan, and theformer Soviet Union (Inoue, M., et al., Zentralbl Bakteriol MikrobiolHyg [B], 186, 504-511, (1988), Nuorti, J. P., et al., J Infect Dis, 189,766-774, (2004), Rodina, L. V., et al., Zh Mikrobiol EpidemiolImmunobiol, 116-118, (1998), Toyokawa, Y., et al., Kansenshogaku Zasshi,67, 36-44, (1993)). Most Y. pseudotuberculosis infections are assumed tobe transmitted by the oral-fecal route; however, a vehicle oftransmission has not been identified in many cases. In the UnitedStates, infections by Y. enterocolitica are more common than those withY. pseudotuberculosis, and are typically associated with the consumptionof contaminated pork products (Ray, S. M., et al., Clin Infect Dis, 38Suppl 3. S181-189, (2004)). The incidence of human disease caused by theY. enterocolitica in the U.S. is difficult to determine, simply becauseinfections associated with this organism are typically self-limiting andinsufficient detection techniques have limited the ability to correctlydiagnose the causative agent. However, FoodNet surveillance for1996-1999 estimated approximately 1 case of Y. enterocolitica infectionper 100,000 in the United States (Ray, S. M., et al., Clin Infect Dis,38 Suppl 3, S181-189, (2004)).

Plague is an infectious disease of animals and humans having bothenzootic and epizootic components of transmission. The most naturallyoccurring means of transmission is from an infected rodent reservoir tofleas, which serve as natural vectors for transmission to humans.However, human-to-human transmission can also occur by direct contact orrespiratory inhalation of contaminated droplets (Pneumonic form).Nevertheless, in natural infections Y. pestis typically enter humans bya subcutaneous route into the bloodstream, where they travel to thelymph nodes and begin to multiply. Clinical manifestations of plagueinclude large swollen masses near the lymph nodes, referred to as bubos.Occasionally, Y. pestis multiplies rapidly in the bloodstream, inducingsepticemia with an accompanying general malaise that includes fever,headache, chills, and occasionally gastrointestinal disturbances. Thesesymptoms are often misdiagnosed early, and antibiotic therapy maytherefore be administered too late for effective intervention.Septicemic infection by Y. pestis has a 50% fatality rate (Perry, R. D.and J. D. Fetherston, Clin Microbiol Rev, 10, 35-66, (1997)), and canlead to pulmonary infection. The pneumonic form of plague is extremelyinfectious by the aerosol route and is characterized by a rapid onset ofdisease and a mortality rate close to 100%. Therefore, althoughantibiotic therapies are available and effective if administered early,the rapid onset of pneumonic plague and the misdiagnosis of septicemicplague are major obstacles in treatment of the disease.

Y. enterocolitica and Y. pseudotuberculosis are consideredenteropathogens since most human infections are transmitted by thefecal-oral route and are limited to the gastrointestinal tract. In anormal host, Y. enterocolitica causes a diarrheal illness, which may beaccompanied by fever and lower quadrant pain that mimics appendicitis.Y. pseudotuberculosis typically does not cause diarrheal illness, and ismore likely to cause mesenteric lymphadenitis which can be misdiagnosedas appendicitis. Following ingestion, both organisms attach to theintestinal lymphoid tissues and traverse the mucosal layer, where theycan subsequently multiply in the mesenteric lymph nodes and migrate tothe spleen and liver (Lian, C. J., et al., J Med Microbiol, 24, 219-226,(1987), Une, T., Microbiol Immunol, 21, 505-516, (1977)). Y.pseudotuberculosis and some serotypes of Y. enterocolitica can alsospread to the vascular system and cause fatal cases of septicemia(Bottone, E. J., Clin. Microbiol. Rev., 10, 257-276, (1997), Lenz, T.,et al., J Infect Dis, 150, 963, (1984)), although these more invasiveinfections are typically limited to susceptible individuals. Y.enterocolitica has also been associated with septicemia following bloodtransfusions; in these cases, the blood supply was contaminated with theorganism, which can survive and grow at refrigeration temperatures(Natkin, J. B., KG, Clin Lab Med, 19, 523-536, (1999)). Furthermore,intestinal Yersinia infections can lead to delayed sequelae such asreactive arthritis and thyroiditis (Bottone, E. J., Clin. Microbiol.Rev., 10, 257-276, (1997), Gaston, J. S., et al., Arthritis Rheum., 42,2239-2242, (1999), Taccetti, G., et al., Clin Exp Rheumatol, 12,681-684, (1994)). Antibiotic therapy has not been demonstrated to reducethe severity or duration of gastrointestinal illness caused by these twopathogens (Hoogkamp-Korstanje, J., J Antimicrob Chemother, 20, 123,(1987), Pai, C. H., et al., J Pediatr, 104, 308-11, (1984)). However, asusceptible host is typically treated with antibiotics to prevent moreserious clinical manifestations of disease. Septicemia caused by eitherof these enteropathogens is also generally treated with antibiotics, andsuch therapies are frequently successful against Y. enterocolitica(Gayraud, M., et al., Clin Infect Dis, 17, 405-10, (1993)). In contrast,antibiotic therapy has traditionally been less effective in patientswhere septicemia is caused by Y. pseudotuberculosis, and the mortalityrate associated with Y. pseudotuberculosis septicemia is approximately75% (Natkin, J. B., KG, Clin Lab Med, 19, 523-536, (1999)).

Although natural infection by Y. pestis is rare in this country, thereis fear that the organism will become a bioterrorism agent. As a tool ofdeliberate mass infection, the Y. pestis organism is a prime candidatedue to several characteristics. First, the organism is highly infectiouswhen spread by aerosol, a convenient method of mass dissemination.Second, there is a high mortality rate associated with Y. pestisinfection if left untreated, and the pneumonic form of plague isdistinguished by a rapid onset of symptoms that may be recognized toolate for an effective intervention. Finally, Y. pestis has awell-defined genetic system, thus antibiotic-resistant strains arerelatively easy to engineer.

Several plague vaccines with varying levels of efficacy and safety havebeen investigated. One of the earliest vaccines consisted of killedwhole cells (KWC) of Y. pestis; this type of vaccine was first used inthe late 1890's and confers protection against the bubonic form ofplague. However, there is evidence that KWC immunizations offer littleprotection against pneumonic plague (Cohen, R. J. and J. L. Stockard,JAMA, 202, 365-366, (1967), Meyer, K. F., Bull World Health Organ, 42,653-666, (1970)), and an additional drawback to these vaccines is thatmultiple injections over several months are required for protectiveimmunity. An attenuated strain of Y. pestis, strain EV76, has beenstudied as a live vaccine for plague. In mouse studies, this vaccine hasbeen shown to protect against both subcutaneous and inhalationchallenges and requires as few as one dose for protection (Russell, P.,et al., Vaccine, 13, 155-1556, (1995)). However, strain EV76 is notfully avirulent, causing death in approximately 1% of vaccinated mice(Russell, P., et al., Vaccine, 13, 1551-1556, (1995)). Interestingly,there have been several unsuccessful attempts to create an avirulentstrain of Y. pestis suitable for use as a live vaccine (Titball, R. W.and E. D. Williamson, Vaccine, 19, 4175-4184, (2001)).

Subunit vaccines are considered to be the most promising type of vaccinefor safe and effective prevention of plague, primarily because there isno fear of adverse effects in a human host. Several surface proteinsassociated with Yersinia virulence were tested for their immunogenicity;all of these proteins induced an antibody response but only the F1capsule and the secreted V antigen elicited good protection againstchallenge (Titball, R. W. and E; D. Williamson, Vaccine, 19, 4175-4184,(2001)). Both F1 and V antigen provide protection as individual antigensin animal models, although the combination of the two antigens providessuperior protection. Many recent studies have tested F1/V vaccinesformulated with alternative adjuvants in an attempt to find the bestdelivery system for the F1 and V antigens (Alpar, H. O., et al., Adv.Drug Deliv. Rev., 51, 173-201, (2001), Byles, J. E., et al., J ControlRelease, 63, 191-200, (2000), Jones, S. M., et al., Vaccine, 19,358-366, (2001), Reddin, K. M., et al., Vaccine, 16, 761-767, (1998),Williamson, E. D., et al., Vaccine, 19, 566-571, (2000). Williamson, E.D., et al., Vaccine, 14, 1613-9, (1996)).

Other innovative strategies have used attenuated Salmonella strains asvaccine carriers for Y. pestis antigens. When a Salmonella aroA mutantexpressing an F1/V fusion protein was used as a vaccine strain, 86% ofmice survived a subsequent lethal challenge dose of Y. pestis (Leary, S.E., et al., Microb Pathog, 23, 167-179, (1997)). Similarly, a vaccineconsisting of a DNA plasmid bearing a gene encoding truncated-F1 capsuleprovided 80 to 100% protection in different mouse strains (Grosfeld, H.,et al., Infect Immun, 71, 374-383, (2003)). In addition, a group ofinvestigators mapped the B- and T-cell epitopes of the F1 antigen andutilized the immunoreactive peptides in vaccine formulations (Sabhnani,L, et al., FEMS Immunol Med Microbiol, 38, 215-29, (2003)). Theirresults indicated that a mixture of epitopic peptides protected 83% ofmice against a lethal dose of Y. pestis.

In contrast to the extensive search for protective plague vaccines, verylittle research efforts have been focused on preventing infections bythe enteropathogenic Yersinia species. However, a few studies havedemonstrated promising results. For example, attenuated Y.enterocolitica strains administered orally to mice displayed protectiveeffects, reducing the bacterial load in the spleen and liver followingoral challenge (Igwe, E. I., et al., Infect Immun, 67, 5500-5507,(1999)). However, these strains were engineered primarily as live oralvaccine carriers, and no further testing of these strains for preventionof yersiniosis has been reported. Two subunit vaccines were demonstratedas effective in animal models of infection. The first consisted ofcellular extracts from Y. enterocolitica and was administeredintranasally to mice. The immunized mice demonstrated enhanced clearanceof an intranasal challenge dose of Y. enterocolitica from the lungs (DiGenaro, M. S., et al., Microbiol. Immunol., 42, 781-788, (1998)). Asecond subunit vaccine was formulated using a heat shock protein HSP60from Y. enterocolitica adjuvanted with interleukin-12 (Noll, A. andAutenriethlb, Infect Immun, 64, 2955-2961, (1996)). Immunizations withthis vaccine resulted in significantly fewer bacteria in mouse spleensfollowing challenge, illustrating a protective effect. Additional workutilized a vaccine consisting of DNA encoding the Y. enterocoliticaHSP60 in intramuscular immunizations in mice (Noll, A., et al., Eur JImmunol, 29, 986-996, (1999)). This study demonstrated that hsp60 mRNAwas present in various host tissues following immunization, butprotection against Y. enterocolitica challenge was limited to the spleenand no protection was observed in the intestinal mucosa.

The similarities and differences between the diseases caused by thepathogenic Yersinia species have been the focus of much research in thepast decade. This is partly due to several observations that suggest thepathogenic Yersinia provide a useful model of pathogen evolution. First,DNA hybridization studies and recent genomic comparisons of fullysequenced Y. pestis and Y. pseudotuberculosis strains have indicatedthat these two pathogens are highly related (Chain, P. S., et al., Proc.Natl. Acad. Sci. USA, 101, 13826-13831, (2004), Ibrahim, A., et al.,FEMS Microbiol Lett, 114, 173-177, (1993)), and it has been estimatedthat Y. pestis evolved from Y. pseudotuberculosis as recently as 1,500to 20,000 years ago (Achtman, M., et al., Proc. Natl. Acad. Sci. USA,96, 14043-14048, (1999)). However, despite their close evolutionaryrelationship, Y. pseudotuberculosis and Y. pestis cause very differentdiseases in humans. Furthermore, partial sequencing and 16s RNAhybridization studies suggested that Y. enterocolitica is more distantlyrelated to the other pathogenic species of this genus (Ibrahim, A., etal., FEMS Microbiol Lett, 114, 173-177, (1993), Moore, R. L. and R. R.Brubaker, Int J Syst Bacteriol, 25, 336-339, (1975)), although Y.enterocolitica causes gastrointestinal infections similar to thoseobserved with Y. pseudotuberculosis. Recent research has thus beenfocused on the virulence genes of the three pathogenic Yersinia speciesin an attempt to elucidate the different mechanisms they employ to causedisease. Mouse models have been particularly instructive in studyingYersinia pathogenesis, since all three species cause similar diseases inmice when injected intravenously, and more natural infections can beeffectively simulated through oral and pneumonic challenge routes inmice.

A few virulence factors are unique to Y. pestis. These include proteinsencoded on the Y. pestis plasmids pPCP and pMT, plasmids that are notfound in Y. enterocolitica or Y. pseudotuberculosis. The pPCP plasmidencodes the plasminogen activator, a protein involved in rapiddissemination of bacteria into mammalian host tissues followingsubcutaneous injection (Sodeinde, O. A., et al., Science, 258,1004-1007, (1992)). The pMT plasmid harbors at least two genes that aidin the infection of non-human hosts. The pMT-encoded caf1 gene isrequired for assembly of the F1 capsule, a factor that inhibitsphagocytosis in the murine host but is not required for virulence inprimates (Friedlander, A. M., et al., Clin. Infect. Dis., 21 Suppl 2,S178-181, (1995)). The murine toxin is also encoded on the pMT plasmid,and is believed to promote survival in the flea although it is not arequired virulence factor in murine hosts (Hinnebusch, B. J., et al.,Science, 296, 733-735, (2002), Hinnebusch, J., et al., Int J MedMicrobiol, 290, 483-487, (2000)). Other differences between the speciesare the structures of the lipopolysaccharide (LPS) molecules produced bythe yersiniae. Both Y. enterocolitica and Y. pseudotuberculosis expressvariable O-antigen side chains, which have been theorized to enhancesurvival in the gastrointestinal tract (Reeves, P., Trends Microbiol.,3, 381-386, (1995)) and may inhibit complement-mediated lysis duringinvasive disease (Karlyshev, A. V., et al., Infect Immun, 69, 7810-7819,(2001)). In contrast, Y. pestis has a rough LPS phenotype with noO-specific side chains due to mutations in several O-antigenbiosynthesis genes (Prior, J. G., et al., Microb. Pathog., 30, 48-57,(2001), Skurnik, M. P., A; Ervela, E. Mol Microbiol, 37, 316-330,(2000)).

Interestingly, genomic sequencing projects revealed that severalvirulence genes present in all three pathogenic Yersinia species haveacquired mutations in Y. pestis that rendered them non-functional(Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101, 13826-13831,(2004), Parkhill, J., et al., Nature, 413, 523-527, (2001)). Some ofthese encode invasin proteins that function during intestinal invasionin the enteropathogenic Y. enterocolitica and Y. pseudotuberculosisspecies, a host niche not colonized by Y. pestis (Simonet, M., et al.,Infect Immun, 64, 375-379, (1996)). Other genes with lost function in Y.pestis include those involved in intermediary metabolism, and thesefunctional losses are theorized to be part of the evolution of Y. pestisinto an obligate parasitic species with the inability to survive outsidethe host (Parkhill, J., et al., Nature, 413, 523-527, (2001)). Researchon the pathogenesis of Yersinia has largely been focused on the 70 kbvirulence plasmid that is found in all pathogenic species of Yersinia.The sequence of this plasmid, called pYV in Y. pseudotuberculosis andpathogenic Y. enterocolitica and pCD1 in Y. pestis, is remarkablyconserved between Y. pseudotuberculosis and Y. pestis (Chain, P. S., etal., Proc. Nat). Acad. Sci. USA, 101, 13826-13831, (2004)). Accordingly,the more distantly-related Y. enterocolitica species harbors a moredivergent pYV plasmid, but the virulence gene sequences are highlyconserved among all three species (Hu, P., et al., J Bacteriol, 180,5192-5202, (1998), Snellings, N. J., et al., Infect Immun, 69, 4627-38,(2001)). Focus on this plasmid began when experiments determined thatthe pYV plasmid is absolutely required for virulence of Yersinia,although the plasmid alone cannot restore virulence to specificavirulent strains suggesting that non-pVY genes are also involved inpathogenesis (Heesemann, J., et al., Infect Immun, 46, 105-110, (1984),Heesemann, J. and R. Laufs, J Bacteriol, 155.761-767, (1983)). A largelocus on this plasmid encodes the Ysc-Yop system, a type III secretionsystem and its associated effector proteins. This system was the firstexample of a type 11 secretion apparatus, now identified in many animaland plant microbial pathogens (for review, see Cornelis, G. R., Nat.Rev. Mol. Cell. Biol., 3, 742-752, (2002)). The Yersinia Yop-Yscsecretion system includes “injectisome” proteins, translocator effectorproteins, and Yop effector proteins. Electron microscopy and labelingstudies with various type III secretory systems revealed that theinjectisome proteins form a pore spanning the cytoplasmic and outermembranes of the bacteria and project a needle-like structure from thecell surface (Blocker. A., et al., Mol. Microbiol., 39, 652-663, (2001),Kimbrough, T. G. and S. I. Miller, Proc Natl Acad Sci USA, 97,11008-11013, (2000), Kubori, T., et al., Science, 280, 602-605, (1998),Sukhan, A., et al., J Bacteriol, 183, 1159-1167, (2001)). Thetranslocator proteins appear to interact with host macrophages andpolymorphonuclear neutrophils (PMNs), forming a pore-like structure inthe host cell membrane (Neyt, C. and G. R. Cornelis, Mol Microbiol, 33,971-981, (1999)). The assembled secretion apparatus then allows theeffector Yops to be translocated across the bacterial cell membranes andinjected into the host cell, where they function by interfering withvarious immune response pathways (Bleves, S. and G. R. Cornelis,Microbes Infect., 2, 1451-1460, (2000), Cornelis, G. R., Nat. Rev. Mol.Cell. Biol., 3, 742-752, (2002)). The yadA gene is also present on thepYV plasmid, encoding the YadA adhesin with the ability to bind andadhere to eukaryotic cells (Eitel, J. and P. Dcrsch, Infect Immun, 70,4880-91, (2002), Skurnik, M., et al., Infect Immun, 62, 1252-61,(1994)). This protein only appears to be functional in theenteropathogenic Yersinia, as a frameshift mutation in the Y. pestisyadA gene renders it non-functional (Hu, P., et al., J Bacteriol, 180,5192-5202, (1998)).

The involvement of iron in Yersinia infections has long beenestablished. For example, iron-overloaded patients such as thoseafflicted with f-thalassemia are highly susceptible to Yersiniainfections (Farmakis, D., et al., Med. Sci. Monit., 9, RA 19-22,(2003)). Furthermore, virulence could be restored in specific avirulentY. pestis mutants by the addition of heme or heme-containing compounds(Burrows, T. W. and S. Jackson, Br. J. Exp. Pathol., 37, 577-583,(1956)). These early observations with Yersinia and other bacteria ledresearchers to study some of the microbial mechanisms of iron uptake. Inmammalian hosts, available iron is extremely limited; intracellular ironis complexed with storage proteins, and extracellular iron is bound bythe host proteins transferrin and lactoferrin. These iron-restrictedconditions limit the growth of microbial invaders, thus acting as adefense barrier to infection. Many pathogens have evolved the ability toscavenge iron under these iron-poor conditions, effectively “stealing”iron from transferrin or heme-containing compounds. One of the mostcommon mechanisms utilized by bacteria is the synthesis and secretion ofsiderophores, small molecules with a high affinity for iron (Andrews, S.C., et al., FEMS Microbiol. Rev., 27, 215-237, (2003)). Theiron-siderophore complexes are bound by outer membrane receptors on thebacterial cell surface, and through the concerted action of outermembrane, periplasmic, and ABC transporter proteins, iron is transportedinto the cell. Other outer membrane receptors can directly bind heme andheme-containing compounds, scavenging the iron from these molecules. Therole of several Yersinia iron uptake systems has been elucidated, whilemany more putative systems have been identified but not characterized.

Although Yersinia can use various siderophores produced by otherbacteria and fungi to obtain iron, yersiniabactin is the onlyYersinia-produced siderophore that has been detected (Baumler, A., etal., Zentralbl. Bakteriol., 278, 416-424, (1993), Rabsch, W. and G.Winkelmann, Biol Met, 4, 244-250, (1991). Reissbrodt, R. and W. Rabsch,Zentralbl Bakteriol Mikrobiol Hyg [A], 268, 306-317, (1988)). Theyersiniabactin system is encoded by the ybt genes present on thechromosomal high-pathogenicity island (HPI), a locus that is associatedwith highly pathogenic strains of Yersinia (de Almeida, A. M., et al.,Microb. Pathog., 14, 9-21, (1993), Rakin, A., et al., J Bacteriol, 177,2292-2298, (1995)). The ybt genes encode proteins involved in thesynthesis and secretion of the siderophore yersiniabactin (ybtS, irp1,irp2, ybtE, ybtT), as well as the cytoplasmic (ybtP, ybrQ) and outermembrane proteins (psnlfyuA) required for uptake of theiron-yersiniabactin complexes (Carniel, E., Microbes Infect., 3,561-569, (2001)). Mutations in genes for yersiniabactin synthesis and/oruptake resulted in reduced Yersinia virulence in mouse models ofinfection (Bearden. S. W., et al., Infect. Immun., 65, 1659-1668,(1997), Brem, D., et al., Microbiology, 147, 1115-1127, (2001), Rakin,A., et al., Mol Microbiol, 13, 253-263, (1994)), indicating that thissystem is an important virulence factor in Yersinia pathogenesis. Thenucleotide sequence of the ybt genes are at least 97% identical betweenthe three pathogenic Yersinia species (Carniel, E., Microbes Infect., 3,561-569, (2001), Chain, P. S., et al., Proc. Natl. Acad. Sci. USA, 101,13826-13831, (2004)), and the Y. pestis and Y. pseudotuberculosis ybtsystems were demonstrated to be interchangeable (Perry, R. D., et al.,Microbiology, 145 (Pt 5), 1181-1190, (1999)). These analyses indicatedthat the functions of these homologs are likely conserved among thethree species. Furthermore, the HPI has been discovered in variouspathogenic species including some strains of E. coli, Citrobacter, andKlebsiella (Bach, S., et al., FEMS Microbiol. Lett., 183, 289-294,(2000)). The Ybt proteins expressed by these organisms are quitesimilar, indeed, antibodies raised against several of the Yersinia Ybtproteins recognized the corresponding proteins from the other pathogens(Bach, S., et al., FEMS Microbiol. Lett., 183, 289-294, (2000), Karch,H., et al., Infect Immun, 67, 5994-6001, (1999)). These results suggestthat the acquisition of the ybt system is relatively recent among thesepathogens and may have contributed to the invasive phenotypes associatedwith many of these serotypes.

Several additional ybt-independent iron uptake systems have beendetected in Yersinia species based on mutation analysis, homology toknown iron acquisition proteins, or the presence of iron-responsiveregulatory elements. One such regulatory element is the “Fur box,” anucleotide sequence that binds the regulatory protein Fur when it iscomplexed with iron. The binding of Fe-Fur to a Fur box repressestranscription of downstream promoters, and when iron becomes limiting,apo-Fur dissociates from DNA and transcription is derepressed. Fur andits homologs have been found in most species of bacteria, and regulatemany genes in addition to iron uptake systems in diverse organisms(Campoy, S., et al., Microbiology, 148, 1039-1048, (2002), Horsburgh, M.J., et al., Infect Immun, 69, 3744-3754, (2001), Sebastian, S., et al.,J Bacteriol, 184, 3965-3974, (2002), Stojiljkovic, I., et al., J MolBiol, 236, 531-545, (1994)). Analysis of the Y. pestis genome identifiedmany genes with Fur boxes upstream of their respective promoters, mostof which encoded proteins with homology to known iron uptake systems(Panina, E. M., et al., Nucleic Acids Res, 29, 5195-5206, (2001)).Although few of these genes have been studied for function, severalappear to encode iron-siderophore receptor proteins (omrA, irgA, itrA,ihaB, fauA) and iron ABC transporters (irsTUS, itpPTS). Since Yersiniacan utilize siderophores produced by other organisms, these proteins maybe responsible for the “siderophore piracy” observed with Yersinia. Suchmethods of iron acquisition are common among bacterial pathogens.

Several studies have elucidated the functions of other putative ironuptake systems. For example, the Hmu system of Y. pestis wasdemonstrated to acquire iron through the uptake of heme andheme-containing compounds (Hornung, J. M., et al., Mol Microbiol, 20,725-39, (1996)). Although the ability to use heme as an iron sourceseems advantageous for a pathogen, the Y. pestis hmu mutant was fullyvirulent in a mouse model of infection (Thompson, J. M., et al., InfectImmun, 67, 3879-92, (1999)). A second putative heme-uptake system wasidentified in Y. pestis on the basis of sequence homology. The has genesof Y. pestis are homologs of the hemophore-dependent heme acquisitiongenes of Pseudomonas and Serratia (Rossi, M. S., et al., Infect Immun,69, 6707-6717, (2001)). In these organisms, a hemophore (HasA) issecreted that binds heme and delivers it to bacterial surface receptors(HasR) to transport heme into the cell. The Y. pestis HasA protein wasdetermined to be Fur-regulated, secreted, and capable of binding heme.However, a mutation in these genes had no effect on virulence in themouse, even when a double mutant was tested (Rossi, M. S., et al.,infect Immun, 69, 6707-6717, (2001)). Therefore, the roles of theputative heme uptake systems in pathogenesis remain elusive, and mayindicate that heme uptake is more important during infection ofnon-murine hosts.

The functions of two putative iron ABC transport systems have also beenstudied in Yersinia. The Yfe system can transport iron and manganese inY. pestis, and yfe mutants demonstrated reduced virulence in mousemodels of infection (Bearden, S. W. and R. D. Perry, Mol. Microbiol.,32, 403-414, (1999)). The second putative iron ABC transporter proteinsare encoded by the yfu genes, identified by the presence of an upstreamFur box (Gong, S., et al., Infect. Immun., 69, 2829-2837, (2001)). Whenexpressed in E. coli, the yfu genes restored growth in iron-poor media;however, comparable studies in Y. pestis failed to determine a role forYfu in iron acquisition, and the yfu-mutant showed no defect in mousevirulence (Gong, S., et al., Infect. Immun., 69, 2829-2837, (2001)).

SUMMARY OF THE INVENTION

The present invention provides a composition including two isolatedpolypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or acombination thereof, and two isolated polypeptides having molecularweights of 40 kDa, 38 kDa, or 37 kDa, or a combination thereof, whereinmolecular weight is determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel. The polypeptides having a molecular weightof 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersiniaenterocolitica when incubated in media containing an iron chelator andnot isolatable when grown in the media without the iron chelator. Insome aspects, the composition may include two different 83 kDapolypeptides isolatable from a Y. enterocolitica when incubated in mediacomprising an iron chelator. The composition protects a mouse againstchallenge with Y. enterocolitica ATCC strain 27729. The composition canfurther include a pharmaceutically acceptable carrier. The polypeptidesmay be isolatable, or in some aspects isolated from Y. enterocolitica isATCC strain 27729. The composition may further include an isolatedpolypeptide having a molecular weight of 268 kDa, 92 kDa, 79 kDa, 54kDa, 45 kDa, 31 kDa, 28 kDa, or a combination thereof, and isolatablefrom a Y. enterocolitica when grown in the media without the ironchelator.

The present invention also provides a composition including two isolatedpolypeptides having molecular weights of 83 kDa, 70 kDa, 66 kDa, or acombination thereof, and two isolated polypeptides having molecularweights of 268 kDa, 79 kDa, or 45 kDa, or a combination thereof, whereinmolecular weight is determined by electrophoresis on a sodium dodecylsulfate-polyacrylamide gel. The polypeptides having a molecular weightof 83 kDa, 70 kDa, or 66 kDa are isolatable from a Yersiniaenterocolitica when incubated in media comprising an iron chelator andnot isolatable when grown in the media without the iron chelator. Thecomposition protects a mouse against challenge with Y. enterocoliticaATCC strain 27729. The composition can further include apharmaceutically acceptable carrier. The polypeptides may be isolatable,or in some aspects isolated from Y. enterocolitica is ATCC strain 27729.

The present invention further provides a composition including isolatedpolypeptides having molecular weights of 268 kDa, 92 kDa, 83 kDa 0.79kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, and28 kDa, wherein molecular weight is determined by electrophoresis on asodium dodecyl sulfate-polyacrylamide gel. The polypeptides areisolatable from a Yersinia enterocolitica, and the composition protectsa mouse against challenge with Y. enterocolitica ATCC strain 27729. Thepolypeptides may be isolatable, or in some aspects isolated from Y.enterocolitica is ATCC strain 27729.

The present invention provides a composition including two isolatedpolypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa,or 64 kDa, or a combination thereof, and two isolated polypeptideshaving molecular weights of 46 kDa, 37 kDa, or a combination thereof,wherein molecular weight is determined by electrophoresis on a sodiumdodecyl sulfate-polyacrylamide gel. The polypeptides having a molecularweight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64 kDa are isolatable froma Yersinia pestis when incubated in media comprising an iron chelatorand not isolatable when grown in the media without the iron chelator.The composition protects a mouse against challenge with Y. pestis strainKIM6+. The composition can further include a pharmaceutically acceptablecarrier. The polypeptides may be isolatable, or in some aspects isolatedfrom Y. enterocolitica is ATCC strain 27729. The composition may furtherinclude an isolated polypeptide having a molecular weight of 254 kDa, 46kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa, and isolatable from a Y.pestis when grown in the media without the iron chelator. Thepolypeptides may be isolatable, or in some aspects isolated from Y.pestis strain KIM6+.

The present invention also provides a composition including two isolatedpolypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa,or 64 kDa, or a combination thereof, and two isolated polypeptideshaving molecular weights of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28kDa, 20 kDa, or a combination thereof, wherein molecular weight isdetermined by electrophoresis on a sodium dodecyl sulfate-polyacrylamidegel. The polypeptides having a molecular weight of 94 kDa, 88 kDa, 77kDa, 73 kDa, or 64 kDa, are isolatable from a Yersinia pestis whenincubated in media comprising an iron chelator and not isolatable whengrown in the media without the iron chelator. The composition protects amouse against challenge with Y. pestis strain KIM6+. The composition canfurther include a pharmaceutically acceptable carrier. The polypeptidesmay be isolatable, or in some aspects isolated from Y. pestis strainKIM6+.

The present invention further provides a composition including isolatedpolypeptides having molecular weights of 254 kDa, 104 kDa, 99 kDa, 94kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 60 kDa, 46 kDa, 44 kDa, 37 kDa, 36kDa, 31 kDa, 28 kDa, and 20 kDa wherein molecular weight is determinedby electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. Thepolypeptides are isolatable from a Yersinia pestis, and the compositionprotects a mouse against challenge with Y. pestis strain KIM6+. Thepolypeptides may be isolatable, or in some aspects isolated from Y.pestis strain KIM6+.

The present invention provides a method for treating in infection in asubject including administering an effective amount of a composition ofthe present invention to a subject having or at risk of having aninfection caused by a Yersinia spp. The subject may be an animal, suchas a fish or a mammal, such as a human. The Yersinia spp. may be, forexample, Y. enterocolitica or Y. pestis, or Y. ruckeri.

The present invention also provides a method for treating a symptom in asubject including administering an effective amount of a composition ofthe present invention to a subject having an infection caused by aYersinia spp. The subject may be an animal, such as a fish or a mammal,such as a human. The Yersinia spp. may be, for example, Y.enterocolitica or Y. pestis, or Y. ruckeri. The symptom may be, forexample, diarrhea, enteritis, plague, red mouth disease, or acombination thereof.

The present invention further provides for treating in infection in asubject including administering an effective amount of a composition toa subject having or at risk of having an infection caused by a Yersiniaspp., wherein the composition includes antibody that specifically bindsa polypeptide of the present invention. The antibody may be polyclonalor monoclonal. In one example, the antibody specifically binds twoisolated polypeptides having molecular weights of 83 kDa, 70 kDa, 66kDa, or a combination, thereof, wherein the polypeptides are isolatablefrom a Yersinia enterocolitica when incubated in media comprising aniron chelator and not isolatable when grown in the media without theiron chelator. In another example, the antibody specifically binds twoisolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77kDa, 73 kDa, or 64 kDa, or a combination thereof, wherein thepolypeptides are isolatable from a Yersinia pestis when incubated inmedia comprising an iron chelator and not isolatable when grown in themedia without the iron chelator.

The present invention also provides a method for treating a symptom in asubject including administering an effective amount of a composition toa subject having an infection caused by a Yersinia spp., wherein thecomposition includes antibody that specifically binds a polypeptide ofthe present invention. The antibody may be polyclonal or monoclonal. Inone example, the antibody specifically binds two isolated polypeptideshaving molecular weights of 83 kDa, 70 kDa, 66 kDa, or a combinationthereof, wherein molecular weight is determined by electrophoresis on asodium dodecyl sulfate-polyacrylamide gel, wherein the polypeptides areisolatable from a Yersinia enterocolitica when incubated in mediacomprising an iron chelator and not isolatable when grown in the mediawithout the iron chelator. In another example, the antibody specificallybinds two isolated polypeptides having molecular weights of 94 kDa, 88kDa, 77 kDa, 73 kDa, or 64 kDa, or a combination thereof, wherein thepolypeptides are isolatable from a Yersinia pestis when incubated inmedia comprising an iron chelator and not isolatable when grown in themedia without the iron chelator.

The present invention further provides kits for detecting antibody thatspecifically binds a polypeptide of the present invention. The kitincludes an isolated polypeptide of the present invention, and a reagentthat detects an antibody that specifically binds the polypeptide. Thepolypeptide and the reagent are typically present in separatecontainers. In one example, the polypeptide may have a molecular weightof 83 kDa, 70 kDa, or 66 kDa, or a combination thereof, wherein thepolypeptide is isolatable from a Yersinia enterocolitica when incubatedin media comprising an iron chelator and not isolatable when grown inthe media without the iron chelator. In another example, the polypeptidemay have a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, or 64kDa, or a combination thereof, wherein the polypeptide is isolatablefrom a Yersinia pestis when incubated in media comprising an ironchelator and not isolatable when grown in the media without the ironchelator.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Detergent-insoluble protein-enriched extracts of Y.enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ resolved byelectrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel. Thenumbers to the left of the gel image denote the molecular weights in kDaof the standards shown in Lane 1. Lane 1, molecular weight standards;Lane 2, Y. pestis strain KIM6+ grown in media supplemented with 300 μMFeCl₃; Lane 3, Y. pestis strain KIM6+ grown in media supplemented with160 μM 2,2-diprydyl; Lane 4, Y. enterocolitica ATCC strain 27729 grownin media supplemented with 160 μM 2,2-diprydyl; Lane 5, Y.enterocolitica ATCC strain 27729 grown in media supplemented with 300 μMFeCl₃.

FIG. 2. Survival of vaccinated and non-vaccinated mice followingchallenge with Y. enterocolitica. Chart showing survival analysis ofmice following immunization with membrane proteins derived from Y.enterocolitica strain 27729 grown under iron-limiting conditions andsubsequent live challenge with strain 27729. Mortality was recorded for7 days following challenge.

FIG. 3. Nucleotide sequences of SEQ ID NOs: 1-23.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides polypeptides and compositions includingpolypeptides. As used herein, “polypeptide” refers to a polymer of aminoacids linked by peptide bonds. Thus, for example, the terms peptide,oligopeptide, protein, and enzyme are included within the definition ofpolypeptide. This term also includes post-expression modifications ofthe polypeptide, for example, glycosylations, acetylations,phosphorylations, and the like. The term polypeptide does not connote aspecific length of a polymer of amino acids. A polypeptide may beisolatable directly from a natural source, or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. In the case of apolypeptide that is naturally occurring, such a polypeptide is typicallyisolated. An “isolated” polypeptide is one that has been removed fromits natural environment. For instance, an isolated polypeptide is apolypeptide that has been removed from the cytoplasm or from the outermembrane of a cell, and many of the polypeptides, nucleic acids, andother cellular material of its natural environment are no longerpresent. An “isolatable” polypeptide is a polypeptide that could beisolated from a particular source. A “purified” polypeptide is one thatis at least 60% free, preferably at least 75% free, and most preferablyat least 90% free from other components with which they are naturallyassociated. Polypeptides that are produced outside the organism in whichthey naturally occur, e.g., through chemical or recombinant means, areconsidered to be isolated and purified by definition, since they werenever present in a natural environment. As used herein, a “polypeptidefragment” refers to a portion of a polypeptide that results fromdigestion of a polypeptide with a protease. Unless otherwise specified,“a,” “an,” “the,” and “at least one” are used interchangeably and meanone or more than one. The terms “comprises” and variations thereof donot have a limiting meaning where these terms appear in the descriptionand claims.

A polypeptide of the present invention may be characterized by molecularweight, mass fingerprint, or the combination thereof. The molecularweight of a polypeptide, typically expressed in kilodaltons (kDa), canbe determined using routine methods including, for instance, gelfiltration, gel electrophoresis including sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis,mass spectrometry, and liquid chromatography including HPLC. Preferably,molecular weight is determined by resolving a polypeptide using an SDSpolyacrylamide gel having a stacking gel of about 4% and a resolving gelof about 10% under reducing and denaturing conditions. Unless indicatedotherwise, molecular weight refers to molecular weight as determined bySDS-PAGE. As used herein, a “mass fingerprint” refers to a population ofpolypeptide fragments obtained from a polypeptide after digestion with aprotease. Typically, the polypeptide fragments resulting from adigestion are analyzed using a mass spectrometric method. Eachpolypeptide fragment is characterized by a mass, or by a mass (m) tocharge (z) ratio, which is referred to as an “m/z ratio” or an “m/zvalue”. Methods for generating a mass fingerprint of a polypeptide areroutine. An example of such a method is disclosed in Example 9.

Polypeptides of the present invention may be metal regulatedpolypeptides. As used herein, a “metal regulated polypeptide” is apolypeptide that is expressed by a microbe at a greater level when themicrobe is grown in low metal conditions compared to growth of the samemicrobe in high metal conditions. Low metal and high metal conditionsare described herein. For instance, one class of metal regulatedpolypeptide produced by Yersinia spp. is not expressed at detectablelevels during growth of the microbe in high metal conditions but isexpressed at detectable levels during growth in low metal conditions.Examples of such metal regulated polypeptides isolatable from Yersiniaenterocolitica have molecular weights of 83 kDa, 70 kDa, or 66 kDa. Insome aspects, Y. enterocolitica may produce two different polypeptideseach having a molecular weight of 83 kDa and each expressed atdetectable levels during growth of the microbe in low metal conditionsand not expressed at detectable levels during growth in high metalconditions. Examples of such metal regulated polypeptides isolatablefrom Yersinia pestis have molecular weights of 94 kDa, 88 kDa, 77 kDa,73 kDa, or 64 kDa.

Another type of metal regulated polypeptide produced by Yersinia spp. isexpressed at detectable levels during growth of the microbe in highmetal conditions but significantly more of the polypeptide is expressedduring growth in low metal conditions. The expression of suchpolypeptides is referred to herein as “enhanced” during growth in lowmetal conditions. Typically, the expression of a polypeptide duringgrowth in low metal conditions is at least 10% or at least 50% greaterthan the expression of the polypeptide during growth in high metalconditions. Examples of metal regulated polypeptides showing enhancedexpression and isolatable from Y. enterocolitica have molecular weightsof 268 kDa, 79 kDa, or 45 kDa. Examples of metal regulated polypeptidesshowing enhanced expression and isolatable from Y. pestis have molecularweights of 254 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa.In some aspects, Y. pestis may produce two different polypeptides eachhaving a molecular weight of 31 kDa and each showing enhancedexpression.

The expression of some polypeptides of the present invention is notsignificantly influenced by the presence of a metal. Examples of suchpolypeptides isolatable from Y. enterocolitica have molecular weights of92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or 28 kDa. In someaspects, Y. enterocolitica may produce two different polypeptides eachhaving a molecular weight of 31 kDa and each not significantlyinfluenced by the presence of a metal. Examples of such polypeptidesisolatable from Y. pestis have molecular weights of 104 kDa, 99 kDa, 60kDa, or 44 kDa.

Whether a polypeptide is a metal regulated polypeptide or not can bedetermined by methods useful for comparing the presence of polypeptides,including, for example, gel filtration, gel electrophoresis includingsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),capillary electrophoresis, mass spectrometry, and liquid chromatographyincluding HPLC. Separate cultures of a microbe are grown under highmetal conditions and under low metal conditions, polypeptides of thepresent invention are isolated as described herein, and the polypeptidespresent in each culture are resolved and compared. Typically, an equalamount of polypeptides from each culture is used. Preferably, thepolypeptides are resolved using an SDS polyacrylamide gel having astacking gel of about 4% and a resolving gel of about 10% under reducingand denaturing conditions. For instance, 30 micrograms (μg) of totalpolypeptide from each culture may be used and loaded into wells of agel. After running the gel and staining the polypeptides with CoomasieBrilliant Blue, the two lanes can be compared. When determining whethera polypeptide is or is not expressed at a detectable level, 30 μg oftotal polypeptide from a culture is resolved on an SDS-PAGE gel andstained with Coomasie Brilliant Blue using methods known in the art. Apolypeptide that can be visualized by eye is considered to be expressedat a detectable level, while a polypeptide that cannot be visualized byeye is considered to be not expressed at a detectable level.

Polypeptides of the present invention may have immunogenic activity.“Immunogenic activity” refers to the ability of a polypeptide to elicitan immunological response in an animal. An immunological response to apolypeptide is the development in an animal of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunogenic activity may be protective.“Protective immunogenic activity” refers to the ability of a polypeptideto elicit an immunological response in an animal that prevents orinhibits infection by Yersinia spp., for instance, Y. enterocolitica orY. pestis. Whether a polypeptide has protective immunogenic activity canbe determined by methods known in the art, for instance as described inexample 4 or example 7. For example, a polypeptide of the presentinvention, or combination of polypeptides of the present invention,protect a rodent such as a mouse against challenge with a Yersinia spp.A polypeptide of the present invention may have seroreactive activity.“Seroactive activity” refers to the ability of a candidate polypeptideto react with antibody present in convalescent serum from an animalinfected with a Yersinia spp., preferably Y. enterocolitica or Y.pestis. Polypeptides of the present invention may have immunoregulatoryactivity. “Immunoregulatory activity” refers to the ability of apolypeptide to act in a nonspecific manner to enhance an immune responseto a particular antigen. Methods for determining whether a polypeptidehas immunoregulatory activity are known in the art.

A polypeptide of the present invention has the characteristics of apolypeptide expressed by a reference microbe. The characteristicsinclude both molecular weight and mass fingerprint. The referencemicrobe can be Y. enterocolitica, Y. pseudotuberculosis, Y. pestis, Y.ruckeri, Y. rohdei, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y.intermedia, Y. kristensenii, or Y. moolaretti, preferably Y.enterocolitica, for instance, Y. enterocolitica ATCC strain 27729, or Y.pestis, for instance, Y. pestis strain KIM6+(Gong et al., Infect.Immun., 69:2829-2837 (2001)).

When the reference microbe is Y. enterocolitica, for instance. Y.enterocolitica ATCC strain 27729, a candidate polypeptide is consideredto be a polypeptide of the present invention if it has a molecularweight of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, or 45 kDa, and has amass fingerprint that is similar to the mass fingerprint of a metalregulated polypeptide expressed by the reference microbe and having amolecular weight of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, or 45 kDa,respectively. Preferably, such polypeptides are metal regulated. Forinstance, a candidate polypeptide is a polypeptide of the presentinvention if it has a molecular weight of 83 kDa and has a massfingerprint similar to the mass fingerprint of one of the metalregulated 83 kDa polypeptides produced by the reference strain Y.enterocolitica ATCC strain 27729. A candidate polypeptide is alsoconsidered to be a polypeptide of the present invention if it has amolecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31 kDa, or28 kDa and has a mass fingerprint that is similar to the massfingerprint of a polypeptide expressed by the reference microbe andhaving a molecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31kDa, or 28 kDa, respectively.

When the reference microbe is Y. pestis, for instance, Y. pestis strainKIM6+, a candidate polypeptide is considered to be a polypeptide of thepresent invention if it has a molecular weight of 254 kDa, 94 kDa, 88kDa, 77 kDa, 73 kDa, 64 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or20 kDa, and has a mass fingerprint that is similar to the massfingerprint of a metal regulated polypeptide expressed by the referencemicrobe and having a molecular weight of 254 kDa, 94 kDa, 88 kDa, 77kDa, 73 kDa, 64 kDa, 46 kDa, 37 kDa, 36 kDa, 31 kDa, 28 kDa, or 20 kDa,respectively. Preferably, such polypeptides are metal regulated. Forinstance, a candidate polypeptide is a polypeptide of the presentinvention if it has a molecular weight of 94 kDa and has a massfingerprint similar to the mass fingerprint of one of the metalregulated 94 kDa polypeptides produced by the reference strain Y. pestisstrain KIM6+. A candidate polypeptide is also considered to be apolypeptide of the present invention if it has a molecular weight of 104kDa, 99 kDa, 60 kDa, or 44 kDa and has a mass fingerprint that issimilar to the mass fingerprint of a polypeptide expressed by thereference microbe and having a molecular weight of 104 kDa, 99 kDa, 60kDa, or 44 kDa, respectively.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram negative microbe, more preferably, amember of the family Enterobacteriaceae preferably, a member of thegenus Yersinia, such as Y. enterocolitica, Y. pseudotuberculosis, or Y.pestis. A candidate polypeptide may also be produced using recombinant,enzymatic, or chemical techniques.

A candidate polypeptide may be evaluated by mass spectrometric analysisto determine whether the candidate polypeptide has a mass fingerprintsimilar to one of the polypeptides expressed by a reference microbe andreferred to above by molecular weight. Typically, the candidatepolypeptide is isolated, for instance by resolving the candidatepolypeptide by gel electrophoresis and excising the portion of the gelcontaining the candidate polypeptide. Any gel electrophoresis methodthat separates polypeptides based on differing characteristics can beused, including 1 dimensional or 2 dimensional gel electrophoresis, aswell as liquid chromatographic separation based on, for instance,hydrophobicity, pi, or size. The candidate polypeptide is fragmented,for instance by digestion with a protease. Preferably, the proteasecleaves the peptide bond on the carboxy-terminal side of the amino acidlysine and the amino acid arginine, except when the amino acid followingthe lysine or the arginine is a proline. An example of such a proteaseis trypsin. Methods for digesting a polypeptide with trypsin are routineand known in the an. An example of such a method is disclosed in Example9.

Methods for the mass spectrometric analysis of polypeptides are routineand known in the art and include, but are not limited to, matrixassisted laser desorption/ionization time of flight mass spectroscopy(MALDI-TOF MS). Typically, a mixture containing the polypeptidefragments obtained from a candidate polypeptide is mixed with a matrixthat functions to transform the laser energy to the sample and produceionized, preferably monoisotopic, polypeptide fragments. Examples ofmatrices that can be used include, for instance, sinapinic acid orcyano-4-hydroxycinnamic acid. An example of a method for the analysis ofpolypeptides by MALDI-TOF MS is described in Example 9. The ionizedpolypeptide fragments are separated according to their m/z ratio, anddetected to yield a spectrum of m/z ratio versus intensity. The spectrumincludes m/z values that represent the polypeptide fragments derivedfrom the candidate polypeptide. For any given polypeptide, the amount ofeach polypeptide fragment resulting from a trypsin digestion should beequimolar. However, it is known that trypsin digestion is not always100% efficient, for instance, some sites are more efficiently cleaved.Thus, when MALDI-TOF MS is used to determine m/z values, the intensityof each m/z value is typically not identical. Generally, a spectrum hasa background level of noise present across most of the x-axis (i.e., theaxis having the values of the m/z ratios). This background level ofnoise varies depending on the running conditions and the machine used,and is easily identified by visual inspection of the spectrum. An m/zvalue is generally considered to represent a polypeptide fragment whenthe intensity is at least 2 times greater, 3 times greater, or 4 timesgreater than the background level of noise. The spectrum usuallyincludes other mfz values that are artifacts resulting from, forinstance, incomplete digestion, over digestion, other polypeptides thatmay be present in the mixture, or the protease used to digest thepolypeptide including m/z values resulting from autolysis of theprotease. This method of digesting a polypeptide with a protease isrecognized by the art as resulting in a mass fingerprint of greatspecificity that can be used to accurately characterize the polypeptideand distinguish it from other polypeptides.

In this aspect of the invention, when a candidate polypeptide isanalyzed by mass spectroscopy, preferably both the candidate polypeptideand the polypeptide from the reference microbe are prepared and analyzedtogether, thereby decreasing any potential artifacts resulting fromdifferences in sample handling and running conditions. Preferably, allreagents used to prepare and analyze the two polypeptides are the same.For instance, the polypeptide from the reference microbe and thecandidate polypeptide are isolated under substantially the sameconditions, fragmented under substantially the same conditions, andanalyzed by MALDI-TOF MS on the same machine under substantially thesame conditions. A mass fingerprint of a candidate polypeptide isconsidered to be similar to the mass fingerprint of a polypeptide from areference microbe when at least 80%, at least 90%, at least 95%, orsubstantially all of the m/z values present in the spectrum of thereference microbe polypeptide and above the background level of noiseare also present in the spectrum of the candidate polypeptide.

In another aspect, a polypeptide is considered to be a polypeptide ofthe present invention if it has a molecular weight of a referencepolypeptide described in Table 1 or Table 2 and has a mass fingerprintthat includes the population of polypeptide fragments of the referencepolypeptide as listed in Table 1 or Table 2. For instance, a polypeptideof the present invention includes a polypeptide of 83 kDa and a massfingerprint that includes polypeptide fragments having masses of 686.37,975.45, 1000.53, 1015.46, 1140.65, 1169.68, 1170.64, 1197.57, 1342.55,1356.74, 1394.67, 1452.73, 1476.72, 1520.76, 1692.77, 1715.75, 1828.79,1960.91, 2013.02, 2018.95, 2040.97, 2163.05, 2225.03, 2416.19, and3174.44, or a mass fingerprint that includes polypeptide fragmentshaving masses of 1001.49, 1103.57, 1139.57, 1154.51, 1170.49, 1208.59,1213.67, 1337.70, 1452.86, 1567.84, 1633.85, 1650.82, 1659.91, 1708.77,1748.95, 1849.92, 1986.98, 2103.95, 2111.03, 2163.11, 2386.19, 2452.09,2537.34, and 3422.66. The mass fingerprint of a candidate polypeptidecan be determined by a mass spectrometric method, for instance byMALDI-TOF MS. The mass fingerprint of a candidate polypeptide willgenerally have additional polypeptide fragments and therefore additionalm/z values other than those listed for a polypeptide in Table 1 or Table2. Preferably, when the candidate polypeptide is being compared to apolypeptide in Table 1 or Table 2, the candidate polypeptide is obtainedfrom a Y. pestis, Y. pseudotuberculosis, or Y. enterocolitica, morepreferably, Y. enterocolitica or Y. pestis. A candidate polypeptide canbe obtained by growth of a microbe under low metal conditions and thesubsequent isolation of a polypeptide by the processes described herein.

It is well known in the art that modifications of amino acids can beaccidentally introduced during sample handling, such as oxidation, andformation of carbamidomethyl derivatives. Further, these types ofmodifications alter the m/z value of a polypeptide fragment. Forinstance, if a polypeptide fragment contains a methoinine that isoxidized the m/z value will be increased by 16 relative to the samefragment that does not contain the oxidized methionine. Accordingly,those polypeptide fragments in Tables 1 and 2 having the notation“oxidation (M)” have an m/z value that is increased by 16 relative tothe same fragment that does not contain the oxidized methionine. It isunderstood that the polypeptide fragments of Table 1 and Table 2 can bemodified during sample handling.

TABLE 1 Characteristics of polypeotides obtained from Y. enterocolitica. mass of polypeptide approximate fragmentspredicted amino acid molecular resulting sequence of the  weight infrom  polypeptide fragment   polypeptide kilodaltons trypsin(SEQ ID Numbers listed designation (kDa)¹ digest² in parenthesis) Lw545268 928.45 FHQLDNR (SEQ ID No: 24) 1139.57 VNFTAGVGGYR (SEQ ID No: 25)1311.65 NSVSIGHESLNR (SEQ ID No: 26) 1439.69 ASTSDTGVAVGFNSK(SEQ ID No: 27) 1525.73 SAETLASANVYADSK (SEQ ID No: 28) 1554.69EAFDLSNDALDMAK + Oxidation (M) (SEQ ID No: 29) 1580.77 SAEVLGIANNYTDSK(SEQ ID No: 30) 1595.78 ALGDSAVTYGAGSTAQK (SEQ ID No: 31) 1682.78EAFDLSNDALDMAKK + Oxidation (M) (SEQ ID No: 32) 2109.18AAVAVGAGSIATGVNSVAIGPLSK (SEQ ID No: 33) Lw391A  83 686.37 DIGNIR(SEQ ID No: 34) 975.45 FFVSYQW (SEQ ID No: 35) 1000.53 VNGQDVTLR(SEQ ID No: 36) 1015.46 ASYFDTNAK (SEQ ID No: 37) 1140.65 DLPVSILAGTR(SEQ ID No: 38) 1169.68 QGVLTLVDGIR  (SEQ ID No: 39) 1170.64NIPGLTVTGSGR (SEQ ID No: 40) 1197.57 YYNNSALEPK (SEQ ID No: 41) 1342.55APTMGEMYNDSK (SEQ ID No: 42) 1356.74 IDQIQSLSANLR (SEQ ID No: 43)1394.67 TDDVDGILSFGTR (SEQ ID No: 44) 1452.73 GMTTTVVLGNAFDK(SEQ ID No: 45) 1476.72 IADTMVVTATGNER (SEQ ID No: 46) 1520.76FGSGWLQDEITLR (SEQ ID No: 47) 1692.77 NPQTSAASSTNLMTDR (SEQ ID No: 48)1715.75 FNDLMMAEDDLQFK (SEQ ID No: 49) 1828.79 GSSEGYADVDADKWSSR(SEQ ID No: 50) 1960.91 QEQTPSGATESFPQADIR (SEQ ID No: 51) 2013.02QGTDTGHLNSTFLDPALVK (SEQ ID No: 52) 2018.95 QSDGFNAPNDETISNVLAK(SEQ ID No: 53) 2040.97 VYSAAATGDKSFGLGASAFGR (SEQ ID No: 54) 2163.05LFTDSFASHLLTYGTEAYK (SEQ ID No: 55) 2225.03 VSSSGTPQAGYGVNDFYVSYK(SEQ ID No: 56) 2416.19 GAVSVTPTDWLMLFGSYAQAFR (SEQ ID No: 57) 3174.44SSFEAPMMVTVVEADTPTSETATSATDMLR (SEQ ID No: 58) Lw391B  83 1001.49NDASVQNVR (SEQ ID No: 59) 1103.57 IGFLGQQDAR (SEQ ID No: 60) 1139.57VNLGYAANYR (SEQ ID No: 61) 1154.51 GYGNPSQNYR (SEQ ID No: 62) 1170.49YGDDDQFGVR (SEQ ID No: 63) 1208.59 GHFDTGPITHK (SEQ ID No: 64) 1213.67LLASATWLDPK (SEQ ID No: 65) 1337.70 NVPFNVIGYTSK (SEQ ID No: 66) 1452.86LKPWTRLDLGVR (SEQ ID No: 67) 1567.84 VSLYANHIEALGPGK (SEQ ID No: 68)1633.85 GIELNVFGEPVFGTR (SEQ ID No: 69) 1650.82 TNDTITVVGAQETFR(SEQ ID No: 70) 1659.91 VTPIYGIMVKPWEK  (SEQ ID No: 71) 1708.77NFDSGVPNSAGSLDAMK (SEQ ID No: 72) 1748.95 LYVPYVADSVAGLGGIR(SEQ ID No: 73) 1849.92 VTVDYGSASQVGGALDVGR (SEQ ID No: 74) 1986.98AGGNPLIPTYLDGQVANGGR (SEQ ID No: 75) 2103.95 SEYDVSQNWTVYGSVGASR(SEQ ID No: 76) 2111.03 GYNLDGDDISFGGLFGVLPR (SEQ ID No: 77) 2163.11SGSQYANEANTLKLKPWTR (SEQ ID No: 78) 2386.19 GANAFINGISPSGSGVGGMINLEPK(SEQ ID No: 79) 2452.09 NEETGQYGAPMLTNNNGDATISR (SEQ ID No: 80) 2537.34SAPYQYNGKPVVNAGQIPGIIHSK (SEQ ID No: 81) 3422.66YGGTLALFEITRPTGMVDPATNVYGFYGEQR (SEQ ID No: 82) Lw392  79 836.44 YDTVALR(SEQ ID No: 83) 1017.59 VLLGVDFQK (SEQ ID No: 84) 1070.48 FDDVWSPR(SEQ ID No: 85) 1085.50 SVQATVGYDF (SEQ ID No: 86) 1131.59 ADLGTWAASLK(SEQ ID No: 87) 1188.55 QWADDANTLR (SEQ ID No: 88) 1214.63 VNSQGLELEAR(SEQ ID No: 89) 1235.65 AVPATYYVPAGK (SEQ ID No: 90) 1255.66 LSVIAGYTYNR(SEQ ID No: 91) 1263.65 VPSYTLGDASVR (SEQ ID No: 92) 1360.66 RPQFTSEGHFR(SEQ ID No: 93) 1496.67 GPFDGESNHNVFK (SEQ ID No: 94) 1501.79GAFVQLNVNNIADK (SEQ ID No: 95) 1614.75 WQQIYSYEFSHK (SEQ ID No: 96)1652.77 GFFDGESNHNVFKR (SEQ ID No: 97) 1717.82 GFHGGDVNNTFLDGLR(SEQ ID No: 98) 1770.85 RWQQIYSYEFSHK (SEQ ID No: 99) 1819.86AGHEADLPTSGYTATTTK (SEQ ID No: 100) 1827.01 TDQPLILTAQSVSVVTR(SEQ ID No: 101) 2004.92 DPSGGYHSAVPADGSIYGQK (SEQ ID No: 102) 2066.02GPSSALYGQSIPGGVVMMTSK (SEQ ID No: 103) 2119.91 KYVAACYSTSYCYWGAER(SEQ ID No: l04) 2299.22 YAIAPSLLWQPDENTSLLLR (SEQ ID No: 105) 2307.15LLSDGGSYNVLQVDPWFLER (SEQ ID No: 106) 2782.23 QNASYTHSNTQLEQVYQGGWNSDR(SEQ ID No: 107) 2911.35 LTAGNNNTQVAAFDYTDAISEHWAFR (SEQ ID No: 108)3023.42 RYEQSGVYLQDEMTLDNWHLNLSGR (SEQ ID No: 109) 3286.53QQMDDQNVATVNQALNYTPGVFTGFSGGATR (SEQ ID No: 110) Lw393  70 713.42 VPFVPR(SEQ ID No: 111) 759.42 TVGINR (SEQ ID No: 112) 806.41 YGALMPR(SEQ ID No: 113) 819.42 FDIGGGVR (SEQ ID No: 114) 919.48 GPQGTLYGK(SEQ ID No: 115) 1023.50 GYIEGGVSSR (SEQ ID No: 116) 1051.53 SINYELGTR(SEQ ID No: 117) 1186.57 WNQDVQELR (SEQ ID No. 118) 1199.60 TVDMVFGLYR(SEQ ID No: 119) 1394.68 YGAGSSVNGVIDTR (SEQ ID No: 120) 1436.66LSLSDGSPDPYMR (SEQ ID No: 121) 1479.70 ATQDAYVGWNDIK (SEQ ID No: 122)1540.80 INISVHVDNLFDR (SEQ ID No: 123) 1545.80 TFPSGSLIVNMPQR(SEQ ID No: 124) 1564.76 KLSLSDGSPDPYMR (SEQ ID No: 125) 1667.72SEFTNDSELYHGNR (SEQ ID No: 126) 1730.85 FAPGWSWDINGNVIR (SEQ ID No: 127)1789.81 LAPDDQPWEMGFAASR (SEQ ID No: 128) 1904.85 TYGYMNGSSAVAQVNMGR(SEQ ID No: 129) 1981.02 SAQGGIINIVTQQPDSTPR (SEQ ID No: 130) 1982.93QGTYATLDSSLGWQATER (SEQ ID No: 131) 1995.94 DMQLYSGPVGMQTLSNAGK(SEQ ID No: 132) 2009.89 SSTQYHGSMLGNPFGDQGK (SEQ ID No: 133) 2027.02LAVNLVGPHYFDGDNQLR (SEQ ID No: 134) 2058.99 LRLAPDDQPWEMGFAASR(SEQ ID No: 135) 2132.93 QVDDGDMINPATGSDDLGGTR (SEQ ID No: 136) 2162.17FNLSGPIQDGLLYGSVTLLR (SEQ ID No: 137) 2274.20 VLPGLNIENSGNMLFSTISLR(SEQ ID No: 138) 2363.13 SEFTNDSELYHGNRVPFVPR (SEQ ID No: 139) 2377.30SKFNLSGPIQDGLLYGSVTLLR (SEQ ID No: 140) 2383.07 SNDDQVLGQLSAGYMLTDDWR(SEQ ID No: 141) 2563.22 SASANNVSSTVVSAPELSDAGVTASDK (SEQ ID No: 142)2657.23 YTTDDWVFNLISAWQQQHYSR (SEQ ID No: 143) 2833.50IAQGYKPSGYNIVPTAGLDAKPFVAEK (SEQ ID No: 144) 2929.46SASANNVSSTVVSAPELSDAGVTASDKLPR (SEQ ID No: 145) Lw550  66 867.49VSGLLSHR (SEQ ID No: 146) 881.42 TSEYLNR (SEQ ID No: 147) 883.43 EWHGTVR(SEQ ID No: 148) 1020.59 YTLILVDGK (SEQ ID No: 149) 1086.57 RVDIEVNDK(SEQ ID No: 150) 1167.61 VGKEWHGTVR (SEQ ID No: 151) 1176.69 YTLILVDGKR(SEQ ID No: 152) 1207.63 LMGGVYNVLDK (SEQ ID No: 153) 1345.72IQDSAASISVVTR (SEQ ID No: 154) 1748.72 MDQDENYGTHWTPR (SEQ ID No: 155)1753.77 NEFDFDIGHYVQDR (SEQ ID No: 156) 1850.95 DVPGVVVTGGGSHSDISIR(SEQ ID No: 157) 2520.27 GTRPNSDGSGIEQGWLPPLAAIER (SEQ ID No: 158)2606.16 NNYAITHHGYYDFGNSTSYVQR (SEQ ID No: 159) 2942.50AYTDITDALKDVPGVVVTGGGSHSDISIR (SEQ ID No: 160) 3085.41NGAATFTLTPDDKNEFDFDIGHYVQDR (SEQ ID No: 161) Lw552  45 1139.57VNFTAGVGGYR (SEQ ID No: 162) 1208.61 SSQALAIGSGYR (SEQ ID No: 163)1311.65 NSVSIGHESLNR (SEQ ID No: 164) 1439.69 ASTSDTGVAVGFNSK(SEQ ID No: 165) 1500.74 TTLETAEEHTNKK (SEQ ID No: 166) 1525.73SAETLASANVYADSK (SEQ ID No: 167) 1580.77 SAEVLGIANNYTDSK(SEQ ID No: 168) 1595.78 ALGDSAVTYGAGSTAQK (SEQ ID No: 169) Lw555  37704.42 LGFAGLK (SEQ ID No: 170) 880.43 ADAYSGGLK (SEQ ID No: 171) 970.38DGDQSYMR (SEQ ID No: 172) 121.57 DGNKLDLYGK (SEQ ID No: 173) 1279.54AEDQDQGNPTR (SEQ ID No: 174) 1294.58 VDGLHYFSDDK (SEQ ID No: 175)1334.67 INLLDENEPTK (SEQ ID No: 176) 1509.71 VDGLHYFSDDKSK(SEQ ID No: 177) 1907.96 NAGINTDDIVAVGLVYQF (SEQ ID No: 178) 2245.12NTNFFGLVDGLNFALQYQGK (SEQ ID No: 179) 2324.11 YDANNVYLAATYAQTYNLTR(SEQ ID No: 180) 2642.22 GETQISDQLTGYGQWEYQANLNK (SEQ ID No: 181)2984.54 AQNIELVAQYQFDFGLRPSVAYLQSK (SEQ ID No: 182) 3087.49FGLKGETQISDQLTGYGQWEYQANLNK (SEQ ID No: 183) Lw557  31 863.51 TVYLQIK(SEQ ID No: 184) 1403.71 NTSDKNMLGLAPK + Oxidation (M) (SEQ ID No: 185)1615.81 FEEAQPVLEDQLAK (SEQ ID No: 186) 1779.83 TQMSETIWLEPSSQK +Oxidation (M) (SEQ ID No: 187) 1875.92 VQTSTQTGNKHQYQTR (SEQ ID No: 188)2070.10 VNLKFEEAQPVLEDQLAK (SEQ ID No: 189) 2378.15GYTVTSSPEDAHYWIQANVLK (SEQ ID No: 190) ¹Molecular weight as determinedby SDS-PAGE. ²The mass of a polypeptide fragment can be converted to m/zvalue by adding 1 to the mass. Each mass includes a range of plus orminus 1 Da or plus or minus 300 ppm.

TABLE 2 Characteristics of polypeptides obtained from Y. pestis. mass ofpolypeptide approximate fragments molecular resulting weight in frompredicted amino acid polypeptide kilodaltons trypsin sequence of thedesignation (kDa)¹ digest² polypeptide fragment Lw529 104 643.43 ALISLK(SEQ ID No: 191) 684.36 SIYFR (SEQ ID No: 192) 770.49 ILIGEVK(SEQ ID No: 193) 840.46 NPVARER (SEQ ID No: 194) 898.55 AVQDIILK(SEQ ID No: 195) 961.55 YPLISELK (SEQ ID No: 196) 1136.61 NGIIFSPHPR(SEQ ID No: 197) 1276.63 EAGVQEADFLAK (SEQ ID No: 198) 1292.62NFEEAVEKAEK (SEQ ID No: 199) 1385.65 VVDESEPFAHEK (SEQ ID No: 200)1409.76 NGGLNAAIVGQPATK (SEQ ID No: 201) 1421.84 AAALAAADARIPLAK(SEQ ID No: 202) 1497.82 AVTNVAELNELVAR (SEQ ID No: 203) 1566.75QTAFSQYDRPQAR (SEQ ID No: 204) 1678.39 LLKEFLPASYNEGAK (SEQ ID No: 205)1683.82 YAEIADHLGLSAPGDR (SEQ ID No: 206) 1725.93 GSLPIALEEVATDGAKR(SEQ ID No: 207) 1872.90 EYANFSQEQVDKIFR (SEQ ID No: 208) 1990.01NHFASEYIYNAYKDEK (SEQ ID No: 209) 2020.06 ILINTPASQGGIGDLYKFK(SEQ ID No: 210) 2182.01 EYVEEFDREEEVAAATAPK (SEQ ID No: 211) 2584.21YNANDNPTKQTAFSQYDRPQAR (SEQ ID No: 212) 2842.48AAYSSGKPAIGVGAGNTPVVVDETADIKR (SEQ ID No: 213) Lw530 99 1190.64ILFYTGVNHK (SEQ ID No: 214) 1513.80 YRNIGISAHIDAGK (SEQ ID No: 215)1590.77 HSDDKEPFSALAFK (SEQ ID No: 216) 1596.83 IATDPFVGNLTFFR(SEQ ID No: 217) 1636.82 YLGGEELTEEEIKK (SEQ ID No: 218) 1670.86MEFPEPVISVAVEPK (SEQ ID No: 219) 1713.93 EFIPAVDKGIQEQLK(SEQ ID No: 220) 1718.96 LGANPVPLQLAIGAEEK (SEQ ID No: 221) 1750.91VYSGIVNSGDTVLNSVK (SEQ ID No: 222) 1819.92 EFNVEANVGKPQVAYR(SEQ ID No: 223) 1863.01 EEIKEVHAGDIAAAIGLK (SEQ ID No: 224) 1966.91LHYGSYHDVDSSELAFK (SEQ ID No: 225) 2122.10 VYSGIVNSGDTVLNSVKSQR(SEQ ID No: 226) Lw531 94 961.44 NRDEWSR (SEQ ID No: 227) 1167.49YEYGMFSQK + Oxidation (M) (SEQ ID No: 228) 1257.64 VSVIDENNGRR(SEQ ID No: 229) 1371.63 VLYPDDSTYSGR (SEQ ID No: 230) 1383.64EENDPGLGNGGLGR (SEQ ID No: 231) 1408.71 IIDAPDNNWVPR (SEQ ID No: 232)1520.82 NLDYPSFLLALQK (SEQ ID No: 233) 1668.86 EYADEIWHIKPIR(SEQ ID No: 234) 1685.79 SYVDTQEQVDALYR (SEQ ID No: 235) 1713.78GYGIRYEYGMFSQK + Oxidation (M) (SEQ ID No: 236) 1716.81TLLNIANMGYFSSDR + Oxidation (M) (SEQ ID No: 237) 1796.92TSPFSYTSPVVSVDALK (SEQ ID No: 238) 1832.92 LVEEQYPDDKELLSR(SEQ ID No: 239) 1844.91 KTLLNIANMGYFSSDR + Oxidation (M)(SEQ ID No: 240) 2218.12 IAIHLNDTHPVLSIPEMMR + Oxidation (M)(SEQ ID No: 241) 2426.09 FNQGDYFAAVEDKNHSENVSR (SEQ ID No: 242) Lw532 88888.51 YIQAAVPK (SEQ ID No: 243) 926.46 FNINYTR (SEQ ID No: 244) 945.53SGFLIPNAK (SEQ ID No: 245) 960.54 IGFNIELR (SEQ ID No: 246) 1171.60AQYLYVPYR (SEQ ID No: 247) 1176.57 GLQWQNEFR (SEQ ID No: 248) 1289.64ITGWNAQGQTSK (SEQ ID No: 249) 1332.67 RGLQWQNEFR (SEQ ID No: 250)1357.66 EEQVVEVWNAR (SEQ ID No: 251) 1403.74 IASANQVSTGLTSR(SEQ ID No: 252) 1418.68 FTSVNPTNPEASR (SEQ ID No: 253) 1507.73IYTGPDGTDKNATR (SEQ ID No: 254) 1578.78 FNVSVGQIYYFSR (SEQ ID No: 255)1672.80 QFQVFTAAGNSNAYR (SEQ ID No: 256) 1735.83 TVTATGDVNYDDPQIK(SEQ ID No: 257) 2400.17 LLATHYQQDIPASFADNASNPK (SEQ ID No: 258) 2665.28VYNPDYQQGISQVGTTASWPIADR (SEQ ID No: 259) Lw533 77 686.37 DIGNIR(SEQ ID No: 260) 784.49 RIEIVR (SEQ ID No: 261) 858.41 VSYFDTK(SEQ ID No: 262) 952.50 AKDYISTR (SEQ ID No: 263) 1140.65 DLPVSILAGTR(SEQ ID No: 264) 1155.66 QGVLTLVDGVR (SEQ ID No: 265) 1170.64QVPGLTVTGSGR (SEQ ID No: 266) 1197.57 YYNNSAIEPK (SEQ ID No: 267)1402.71 EQTTEGVKLENR (SEQ ID No: 268) 1408. 68 TDDLDGILSPGTR(SEQ ID No: 269) 1482.73 TALFNWDLAYNR (SEQ ID No: 270) 1522.71EYYTPQGIPQDGR (SEQ ID No: 271) 1550.77 FSSGWLQDEITLR (SEQ ID No: 272)1617.74 HSTDTMVVTATGNER (SEQ ID No: 273) 1674.78 QEQTPGGATESFPQAK(SEQ ID No: 274) 1745.84 KHSTDTMVVTATGNER (SEQ ID No: 275) 1787.92GTWQIDSIQSLSANLR (SEQ ID No: 276) 1819.96 IRFSSGWLQDEITLR(SEQ ID No: 277) 1851.87 VDMQAMTTTSVNIDQAK (SEQ ID No: 278) 1940.75YDNVSGSSDGYADVDADK (SEQ ID No: 279) 2013.02 QGTDTGHLNSTFLDPALVK(SEQ ID No: 280) 2017.97 QSNGFNAPNDETISNVLAK (SEQ ID No: 281) 2056.96VYSSAATGDHSFGLGASAFGR (SEQ ID No: 282) 2168.01 VSSSTPQAGYGVNDFYVSYK(SEQ ID No: 283) 2169.10 LFIESPASHLLTYGTETYK (SEQ ID No: 284) 2426.25TRLFIESPASHLLTYGTETYK (SEQ ID No: 285) 2457.00 YDNYSGSSDGYADVDADKWSSR(SEQ ID No: 286) 2828.33 VSSSTPQAGYGVNDFYVSYKGQEAFK (SEQ ID No: 287)Lw534 73 628.39 IEVIR (SEQ ID No: 288) 748.43 GTIFRR (SEQ ID No: 289)909.42 GGYEDTLR (SEQ ID No: 290) 930.51 TGGLDISIR (SEQ ID No: 291)1291.71 LLDSLALTYGAR (SEQ ID No: 292) 1370.81 LLKNTNIILDSK(SEQ ID No: 293) 1440.70 FTQNYANLSAANK (SEQ ID No: 294) 1478.71YDNSANQLGTIGAR (SEQ ID No: 295) 1586.83 EAAASISVISQNELR (SEQ ID No: 296)1604.86 GMPSAYTLILVDGIR (SEQ ID No: 297) 1640.87 LITNASVPQGSGLAGEK(SEQ ID No: 298) 1654.77 YEYQTTFGGHISPR (SEQ ID No: 299) 1705.82DASRVESSNTGVELSR (SSQ ID No: 300) 1707.83 AYLVWDAQDNWTVK(SEQ ID No: 301) 1757.91 LNWNINEQLSTWLK (SEQ ID No: 302) 1796.97LITNASVPQGSGLAGEKR (SEQ ID No: 303) 1856.01 IREAAASISVISQNELR(SEQ ID No: 303) 1912.94 INSVSIDNTTSTYTNVGK (SEQ ID No: 304) 2004.03DVTLNGAVNNLLDKDFTR (SEQ ID No: 305) 2072.02 FSFYSSGPAVEDQLGLSLR(SEQ ID No: 306) 2155.08 NKINSVSIDNTTSTYTNVGK (SEQ ID No: 307) 2301.07LDFGTWNSSLSYNQTENIGR (SEQ ID No: 308) 2395.11 NYNDLAQALSDVEGVDVNSSTGK(SEQ ID No: 309) 2484.12 AWASSATLEHTFQENTAFGDSSK (SEQ ID No: 310)2557.36 VVYNNLGSEFKPFSVLNLGVAYK (SEQ ID No: 311) 2557.36VVYNNLGSSFKPFSVLNTGVAYK (SEQ ID No: 312) 2675.42TPTLAQLHNGISGVTGQGTITTIGNPK (SEQ ID No: 313)) 2983.33DGIVLANNGDEFAQDAWSLFSEDEWR (SEQ ID No: 314) 3161.51THIFAVGNGTTTAGDVFTSSQSTAGYVVPGR (SEQ ID No: 315) 3184.52ITLGNDNRLDFGTWNSSLSYNQTENIGR (SEQ ID No: 316) 3424.79GGVSTGYKTPTLAQLHNGISGVTGQGTITTIGNPK (SEQ ID No: 317) 3471.62LEPESSVNTEVGVYYENETGFGANVTLFHNR (SSQ ID No: 318) Lw535 64 713.42 VPFVPR(SEQ ID No: 319) 759.42 TVGINTR (SEQ ID No: 320) 773.40 AATLGDAR(SEQ ID No: 321) 806.41 YGALMPR (SEQ ID No: 322) 919.48 GPQGTLYGK(SEQ ID No: 323) 1023.50 GYIEGGVSSR (SEQ ID No: 324) 1051.53 SINYELGTR(SEQ ID No: 325) 1102.55 ADATGVELEAK (SEQ ID No: 326) 1164.56 DMQLYSGPVR(SEQ ID No: 327) 1186.57 WNQDVQELR (SEQ ID No: 328) 1199.60 TVDMVFGLYR(SEQ ID No: 329) 1281.67 TVGINTRIDFF (SEQ ID No: 330) 1394.68YGAGSSVNGVIDTR (SEQ ID No: 331) 1444.73 ADATGVELEAKWR (SEQ ID No: 332)1479.70 ATQDAYVGWNDIK (SEQ ID No: 333) 1545.80 TFPSGSLIVNMPQR(SEQ ID No: 334) 1667.72 SEFTNDSELYHGNR (SEQ ID No: 335) 1692.82ATQDAYVGWNDIKGR (SEQ ID No: 336) 1730.85 FAPGWSWDINGNVIR(SEQ ID No: 337) 1789.81 LAPDDQPWEMGFAASR (SEQ ID No: 338) 1904.85TYGYMNGSSAVAQVNMGR (SEQ ID No: 339) 1968.90 ECTRATQDAYVGWNDIK(SEQ ID No: 340) 1981.02 SAQGGIINIVTQQPDSTPR (SEQ ID No: 341) 2009.89SSTQYHGSMLGNPFGDQGK (SEQ ID No: 342) 2027.02 LAVNLVGPHYFDGDNQLR(SEQ ID No: 343) 2058.99 YETADVTLQAATFYTHTK (SEQ ID No: 344) 2162.17FNLSGPIQDGLLYGSVTLLR (SEQ ID No: 345) 2363.13 SEFTNDSELYHGNRVPFVPR(SEQ ID No: 346) 2377.30 SKFNLSGPIQDGLLYGSVTLLR (SEQ ID No: 347) 2819.49VAQGYKPSGYNIVPTAGLDAKPFVAEK (SEQ ID No: 348) 2929.46SASANNVSSTVVSAPELSDAGVTASDKLPR (SEQ ID No: 349) Lw536 60 1010.51VEDALHATR (SEQ ID No: 350) 1186.65 VAAVKAPGFGDR (SEQ ID No: 351) 1230.66TTLEDLGQAKR (SEQ ID No: 352) 1237.65 ASVEDALHATR (SEQ ID No. 353)1290.65 VGAATEVEMKEK (SEQ ID No: 354) 1566.87 AAVEEGVVAGGGVALIR(SEQ ID No: 355) 1604.88 NVVLDKSFGSPTITK (SEQ ID No: 356) 1620.85SFGSPTITKDGVSVAR (SEQ ID No: 357) 1668.75 QQIEDATSDYDKEK(SEQ ID No: 358) 2020.03 AAHAIAGLKGDNEDQNVGIK (SEQ ID No: 359) 2396.29VVINKDTTIIIDGVGDEAAIQGR (SEQ ID No: 360) Lw537 46 872.51 NLSLLSAR(SEQ ID No: 361) 1000.53 QTVTTPRAQ (SEQ ID No: 362) 1179.55 AAADRDAAYEK(SEQ ID No: 363) 1257.63 NNLDNALESLR (SEQ ID No: 364) 1299.71LSQDLAREQIK (SEQ ID No: 365) 1306.65 DAAYEKINEVR (SEQ ID No: 366)1324.65 AIDSLSYTEAQK (SEQ ID No: 367) 1367.75 TQRPDAVNNLLK(SEQ ID No: 368) 1394.76 YNYLINQLNIK (SEQ ID No: 369) 1435.73ASYDTVLAAEVAAR (SEQ ID No: 370) 1608.93 LKTQRPDAVNNLLK (SEQ ID No: 371)1615.87 FNVGLVAITDVQNAR (SEQ ID No: 372) 1779.96 TILDVLTATTNLYQSK(SEQ ID No: 373) 1951.01 QITGVYYPELASLNVER (SEQ ID No: 374) 1957.97AIDSLSYTEAQKQSVYR (SEQ ID No: 375) 2018.98 QAQYNFVGASELLESAHR(SEQ ID No: 376) 2098.10 SPLLPQLGLSAGYTHANGFR (SEQ ID No: 377) 2177.16QQLADARYNYLINQLNIK (SEQ ID No: 378) 2709.43 INEVRSPLLPQLGLSAGYTHANGFR(SEQ ID No: 379) Lw538 44 775.40 HTPFFK (SEQ ID No: 380) 836.49 EHILLGR(SEQ ID No: 381) 904.49 FAIREGGR (SEQ ID No: 382) 1026.58 AGENVGVLLR(SEQ ID No: 383) 1072.60 GTVVTGRVER (SEQ ID No: 384) 1199.66EGGRTVGAGVVAK (SEQ ID No: 385) 1231.57 ALEGEAEWEAK (SEQ ID No: 386)1232.61 GYRPQFYFR (SEQ ID No: 387) 1289.62 DEGGRHTPPFK (SEQ ID No: 388)1375.63 AFDQIDNAPEEK (SEQ ID No: 389) 1602.76 AFDQIDNAPEEKAR(SEQ ID No: 390) 1613.89 VGEEVEIVGIKDTVK (SEQ ID No: 391) 1709.94LLDEGRAGENVGVLLR (SEQ ID No: 392) 1772.87 GITINTSHVEYDTPAR(SEQ ID No: 393) 1794.95 TKPHVNYGTIGHVDHGK (SEQ ID No: 394) 1904.95ELLSAYDFPGDDLPVVR (SEQ ID No: 395) 1977.01 IIELAGYLDSYIPEPER(SEQ ID No: 396) 2000.01 ARGITINTSHVEYDTPAR (SEQ ID No: 397) Lw683 37690.4064 VGFAGLK (SEQ ID No: 398) 893.4606 ANAYTGGLK (SEQ ID No: 399)910.4330 GNGMLTYR (SEQ ID No: 400) 1049.5617 RANAYTGGLK (SEQ ID No: 401)1114.4931 SSDAAFGFADK (SEQ ID No: 402) 1119.4906 NMSTYVDYK(SEQ ID No: 403) 1121.4697 NGSSSETNNGR (SEQ ID No: 404) 1197.5084NLDGDQSYMR (SEQ ID No: 405) 1262.5567 FADYGSLDYGR (SEQ ID No: 406)1307.6146 IDGLHYFSDNK (SEQ ID No: 407) 1319.7085 INLLDKNDFTK(SEQ ID No: 408) 1422.6739 TTAQNDLQYGQGK (SEQ ID No: 409) 1436.6976YVDIGATYFFNK (SEQ ID No: 410) 1490.6022 AENEDGNHDSFTR (SEQ ID No: 411)1533.8038 GKDIGIYGDQDLLK (SEQ ID No: 412) 1578.7750 TTAQNDLQYGQGKR(SEQ ID No: 413) 2245.1167 NTNFFGLVDGLNFALQYQGK (SEQ ID No: 414)2367.1131 YDANNVYLAANYTQTYNLTR (SEQ ID No: 415) 2487.1124IDGLHYFSDNKNLDGDQSYMR (SEQ ID No: 416) 2684.2718 GETQITDQLTGYGQWEYQVNLNK(SEQ ID No: 417) 2979.5242 AHNIEVVAQYQFDFGLRPSVAYLQSK (SEQ ID No: 418)3292.4764 GVADQNGDGYGMSLSYDLGWGVSASAAMASSLR (SEQ ID No: 419) Lw541 311019.58 ALASNILYR (SEQ ID No: 420) 1074.51 SDPGAAFPWK (SEQ ID No: 421)1202.61 KSDPGAAFPWK (SEQ ID No: 422) 1247.61 IFNLVDENER (SEQ ID No: 423)1321.58 MYNIDYNSFR (SEQ ID No: 424) 1403.64 AWHAGVSYWDGR(SEQ ID No: 425) 1786.80 ALYDAGIGAWYDDETK (SEQ ID No: 426) 1990.03FPDITPVNVVGHSDIAPGR (SEQ ID No: 427) 2090.99 YGYDTSGAVSEVGYNQLIR(SEQ ID No: 428) 2118.12 FPDITPVNVVGHSDIAPGRK (SEQ ID No: 429) Lw542 311142.58 SDPGPLFPWK (SEQ ID No: 430) 1298.68 SDPGPLFPWKR (SEQ ID No: 431)1307.76 AIALQLVPEAQR (SEQ ID No: 432) 1340.64 AWHAGVSSWQGR(SEQ ID No: 433) 1370.68 IPQNGQLDTETR (SEQ ID No: 434) 1578.77GTYQIDTHYPSVAK (SEQ ID No: 435) 1779.95 GAASVAVIQQALAAYGYK(SEQ ID No: 436) 1789.94 FLVLHYTAVGDAESLR (SEQ ID No: 437) 1953.00YNISPSDVVAHSDIAPLR (SEQ ID No: 438) 2190.12 NNLNDTSIGIEIVNLGFTEK(SEQ ID No: 439) 2630.38 AIALQLVPEAQRAWHAGVSSWQGR (SEQ ID No: 440) Lw54420 806.42 LIDGDFK (SEQ ID No: 441) 1113.50 GFESSVDGFK (SEQ ID No: 442)1209.60 VGTWMLGAGYR (SEQ ID No: 443) 1243.58 FSSIFGQSESR(SEQ ID No: 444) 1258.63 YYSVTAGPVFR (SEQ ID No: 445) 1269.60RGFSSSVDGFK (SEQ ID No: 446) 1356.66 VGTWMLGAGYRF (SEQ ID No: 447)1789.94 INEYVSLYGLLGAGHGK (SEQ ID No: 448) 2002.92 YEFNNDWGVIGSFAQTR(SEQ ID No: 449) 2998.43 TSLAYGAGLQFNPHPNFVIDASYEYSK (SEQ ID No: 450)¹Molecular weight as determined by SDS-PAGE. ²The mass of a polypeptidefragment can be converted to m/z value by adding 1 to the mass. Eachmass includes a range of plus or minus 300 ppm.

In yet another aspect, the present invention further includespolypeptides having similarity with an amino acid sequence. Thesimilarity is referred to as structural similarity and is generallydetermined by aligning the residues of the two amino acid sequences(i.e., a candidate amino acid sequence and a reference amino acidsequence) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. Reference amino acid sequencesare disclosed in Table 3 and Table 4. Two amino acid sequences can becompared using commercially available algorithms. Preferably, two aminoacid sequences are compared using the Blastp program of the BLAST 2search algorithm, as described by Tatusova, et al., (FEMS Microbiol Lett1999, 174:247-250), and available through the World Wide Web, forinstance at the internet site maintained by the National Center forBiotechnology Information, National Institutes of Health. Preferably,the default values for all BLAST 2 search parameters are used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and optionally, filter on. In thecomparison of two amino acid sequences using the BLAST search algorithm,structural similarity is referred to as “identities.” Preferably, acandidate amino acid sequence has at least 80% identity, at least 90%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity to a referenceamino acid sequence. Preferably, the molecular weight of the candidateamino acid sequence and the reference amino acid sequence aresubstantially the same value. Preferably, the molecular weight of thecandidate amino acid sequence and the reference amino acid sequence isdetermined by SDS polyacrylamide gel electrophoresis. A candidatepolypeptide can be obtained by growth of a microbe under low metalconditions and the subsequent isolation of a polypeptide by theprocedures disclosed herein.

TABLE 3 NCBI sequence identifier of polypeptide identified by theMolecular weight computer algorithm as having best of reference match tomass fingerprint of polypeptide (kDa)¹ reference polypeptide SEQ ID NO:268 23630568, adhesin YadA 1 83 282049, hemin receptor precursor 2 8349114, ferrichrome receptor FcuA 3 79 565634, ferrioxamine receptor 4 70517386, FyuA precursor 5 66 77958488, Outer membrane receptor 6 forferrienterochelin and colicins 45 23630568, adhesin YadA 7 37 77956419,Outer membrane protein 8 (porin) 31 48605, YlpA protein 9 ¹Molecularweight as determined by SDS-PAGE.

TABLE 4 NCBI sequence identifier of polypeptide identified by theMolecular weight computer algorithm as having best of reference match tomass fingerprint of polypeptide (kDa)¹ reference polypeptide SEQ ID NO:104 22125915, CoA-linked acetaldehyde 10 dehydrogenase 99 51597993,elongation factor G 11 94 15981846, glycogen phosphorylase 12 8845443416, organic solvent tolerance 13 protein precursor 77 22124457,TonB-dependent outer 14 membrane receptor 73 51595142, putativeexogenous ferric 15 siderophore receptor; Iha adhesin 64 22126288,pesticin/yersiniabactin 16 outer membrane receptor 60 51594757,chaperonin GroEL 17 46 22127390, outer membrane channel 18 precursorprotein 44 51597992, elongation factor Tu 19 37 77633559, Outer membraneprotein 20 (porin) 31 22125738, putative regulator 21 31 22125770,putative regulator 22 20 22125223, outer membrane protein X 23¹Molecular weight as determined by SDS-PAGE.

Typically, a candidate amino acid sequence having structural similarityto a reference amino acid sequence has immunogenic activity, protectiveimmunogenic activity, seroactive activity, immunoregulatory activity, ora combination thereof.

The polypeptides expressed by a reference microbe and referred to aboveby molecular weight can be obtained by growth of the reference microbeunder low metal conditions and the subsequent isolation of a polypeptideby the processes disclosed herein. A candidate polypeptide is isolatablefrom a microbe, preferably a gram negative microbe, more preferably, amember of the family Enterobacteriaceae preferably, a member of thegenus Yersinia, such as Y. enterocolitica, Y. pseudotuberculosis, or Y.pestis. A candidate polypeptide may also be produced using recombinant,enzymatic, or chemical techniques.

Also provided by the present invention are whole cell preparations of amicrobe, where the microbe expresses one or more of the polypeptides ofthe present invention. The cells present in a whole cell preparation arepreferably inactivated such that the cells cannot replicate, but theimmunogenic activity of the polypeptides of the present inventionexpressed by the microbe is maintained. Typically, the cells are killedby exposure to agents such as glutaraldehyde, formalin, or formaldehyde.

A composition of the present invention may include at least onepolypeptide described herein, or a number of polypeptides that is aninteger greater than 1 (e.g., at least 2, at least 3, at least 4. Insome aspects, a composition may include at least 2 metal regulatedpolypeptides and at least two polypeptides whose expression is notsignificantly influenced by the presence of a metal. For example, whenthe polypeptides are isolatable from Y. enterocolitica, a compositioncan include 2, 3, 4, 5, or more isolated metal regulated polypeptideshaving molecular weights of 268 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 45kDa, or any subset or combination thereof, and two isolated polypeptideshaving a molecular weight of 92 kDa, 54 kDa, 40 kDa, 38 kDa, 37 kDa, 31kDa, 28 kDa, or any subset or combination thereof. In another example,when the polypeptides are isolatable from Y. pestis, a composition caninclude 2, 3, 4, 5, or more isolated metal regulated polypeptides havingmolecular weights of 254 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 31kDa, 28 kDa, 20 kDa, or any subset or combination thereof, and twoisolated polypeptides having molecular weights of 104 kDa, 99 kDa, 60kDa, 44 kDa, 46 kDa, 37 kDa, 36 kDa, or any subset or combinationthereof. A composition can include polypeptides isolatable from 1microbe, or can be isolatable from a combination of 2 or more microbes.For instance, a composition can include polypeptides isolatable from 2or more Yersinia spp., from 2 or more Y. enterocolitica strains, or froma Yersinia spp. and a different microbe that is not a member of thegenus Yersinia. The present invention also provides compositionsincluding a whole cell preparation of one or more Yersinia spp.

Optionally, a polypeptide of the present invention can be covalentlybound or conjugated to a carrier polypeptide to improve theimmunological properties of the polypeptide. Useful carrier polypeptidesare known in the art. For example, a polypeptide of the presentinvention could be coupled to known Yersinia outer membrane immunogenssuch as the F1 antigen or the V antigen. Likewise, polysaccharidecomponents could be conjugated to the proteins of the present inventionto enhance the protective effect of the compositions. The chemicalcoupling of polypeptides of the present invention can be carried outusing known and routine methods. For instance, various homobifunctionaland/or heterobifunctional cross-linker reagents such asbis(sulfosuccinimidyl) suberate, bis(diazobenzidine), dimethyladipimidate, dimethyl pimelimidate, dimethyl superimidate,disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (see, for instance, Harlow and Lane,Antibodies, A Laboratory Manual, generally and Chapter 5, Cold SpringHarbor Laboratory, Cold Spring Harbor, New York, N.Y. (1988)).

Preferably, such compositions of the present invention include lowconcentrations of lipopolysaccharide (LPS). LPS is a component of theouter membrane of most gram negative microbes (see, for instance.Nikaido and Vaara, Outer Membrane. In: Escherichia coli and Salmonellatyphimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.)American Society for Microbiology, Washington, D.C., pp. 7-22 (1987),and typically includes polysaccharides (0-specific chain, the outer andinner core) and the lipid A region. The lipid A component of LPS is themost biologically active component of the LPS structure and togetherinduce a wide spectrum of pathophysiological effects in mammals. Themost dramatic effects are fever, disseminated intravascular coagulation,complement activation, hypotensive shock, and death. The non-specificimmunostimulatory activity of LPS can enhance the formation of agranuloma at the site of administration of compositions that includeLPS.

The concentration of LPS can be determined using routine methods knownin the art. Such methods typically include measurement of dye binding byLPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189(1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, forinstance, Endotoxins and Their Detection With the Limulus AmebocyteLystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.(1982)). There are four basic commercially available methods that aretypically used with an LAL test: the gel-clot test; the turbidimetric(spectrophotometric) test; the colorimetric test; and the chromogenictest. An example of a gel-clot assay is available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma TechnicalBulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., EastFalmouth, Mass.). Typically, assay conditions include contacting thecomposition with a preparation containing a lysate of the circulatingamebocytes of the horseshoe crab, Limulus polyphemus. When exposed toLPS, the lysate increases in opacity as well as viscosity and may gel.About 0.1 milliliter of the composition is added to lysate. Typically,the pH of the composition is between 6 and 8, preferably, between 6.8and 7.5. The mixture of composition and lysate is incubated for about 1hour undisturbed at about 37° C.. After incubation, the mixture isobserved to determine if there was gelation of the mixture. Gelationindicates the presence of endotoxin. To determine the amount ofendotoxin present in the composition, dilutions of a standardizedsolution of endotoxin are made and tested at the same time that thecomposition is tested. Standardized solutions of endotoxin arecommercially available from, for instance, Sigma Chemical (Catalog No.210-SE). U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503), andAssociates of Cape Cod. Inc., (Catalog No. E0005). In general, when acomposition of the present invention is prepared by isolatingpolypeptides from a microbe by a method as described herein (e.g., amethod that includes disrupting and solubilizing the cells, andcollecting the insoluble polypeptides), the amount of LPS in acomposition of the present invention is less than the amount of LPSpresent in a mixture of the same amount of the microbe that has beendisrupted under the same conditions but not solubilized. Typically, thelevel of LPS in a composition of the present invention is decreased by,in increasing order of preference, at least 50%, at least 60%, at least70%; at least 80%, or at least 90% relative to the level of LPS in acomposition prepared by disrupting, but not solubilizing, the samemicrobe.

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. The compositions of the present invention may beformulated in pharmaceutical preparations in a variety of forms adaptedto the chosen route of administration, including routes suitable forstimulating an immune response to an antigen. Thus, a composition of thepresent invention can be administered via known routes including, forexample, oral; parental including intradermal, transcutaneous andsubcutaneous, intramuscular, intravenous, intraperitoneal, etc., andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, intradermal, transcutaneous and rectallyetc. It is foreseen that a composition can be administered to a mucosalsurface, such as by administration to the nasal or respiratory mucosa(e.g. spray or aerosol), to stimulate mucosal immunity, such asproduction of secretory IgA antibodies, throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in Emery and Straub (WO 01/37810 (2001)), and Emery et al.,(WO 96/01620 (1996)). Implants can be produced at sizes small enough tobe administered by aerosol or spray. Implants also include nanospheresand microspheres.

A composition of the present invention may be administered in an amountsufficient to treat certain conditions as described herein. The amountof polypeptides or whole cells present in a composition of the presentinvention can vary. For instance, the dosage of polypeptides can bebetween 0.01 micrograms (μg) and 300 mg, typically between 0.1 mg and 10mg. When the composition is a whole cell preparation, the cells can bepresent at a concentration of, for instance, 10² bacteria/ml, 10³bacteria/ml, 10⁴ bacteria/ml, 10⁵ bacteria/ml, 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptidesmay be present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 mi. When the composition is a whole cell preparation, the cellsare preferably present in the composition in an amount that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 mi. The amount administered will vary depending on variousfactors including, but not limited to, the specific polypeptides chosen,the weight, physical condition and age of the animal, and the route ofadministration. Thus, the absolute weight of the polypeptide included ina given unit dosage form can vary widely, and depends upon factors suchas the species, age, weight and physical condition of the animal, aswell as the method of administration. Such factors can be determined byone of skill in the art. Other examples of dosages suitable for theinvention are disclosed in Emery et al., (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., a polypeptide or whole cell of the present invention) intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (including, for instance, thoseavailable from under the tradename EMULSIGEN from MVP Laboratories,Ralston, Nebr.). ISA-70, RIBI and other substances known in the art. Itis expected that polypeptides of the present invention will haveimmunoregulatory activity, and that such polypeptides may be used asadjuvants that directly act as T and/or B cell activators or act onspecific cell types that enhance the synthesis of various cytokines oractivate intracellular signaling pathways. Such polypeptides areexpected to augment the immune response to increase the protective indexof the existing composition.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2. IL-4 and/or IL-6. TNF, IFN-alpha.IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include other components known in the art such asan antibiotic, a preservative, an anti-oxidant, or a chelating agent.

The present invention also provides methods for obtaining thepolypeptides described herein. The polypeptides and whole cells of thepresent invention are isolatable from a Yersinia spp. Preferred examplesinclude Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis.Microbes useful for obtaining polypeptides of the present invention andmaking whole cell preparations are readily available. In addition, suchmicrobes are readily isolatable by techniques routine and known in theart. The microbes may be derived from an infected animal as a fieldisolate, and used to obtain polypeptides and/or whole cell preparationsof the present invention, or stored for future use, for example, in afrozen repository at −20° C. to −95° C., in bacteriological mediacontaining 20% glycerol, and other like media.

When a polypeptide of the present invention is to be obtained from amicrobe, the microbe can be incubated under low metal conditions. Asused herein, the phrase “low metal conditions” refers to an environment,typically bacteriological media, which contains amounts of a free metalthat cause a microbe to express or enhance expression of metal regulatedpolypeptides. As used herein, the phrase “high metal conditions” refersto an environment that contains amounts of a free metal that cause amicrobe to either not express one or more of the metal regulatedpolypeptides described herein at a detectable level, or to decreaseexpression of such a polypeptide. Metals are those present in theperiodic table under Groups 1 through 17 (IUPAC notation; also referredto as Groups I-A, II-A, III-B, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B,III-A, IV-A, V-A, VI-A, and VII-A, respectively, under CAS notation).Preferably, metals are those in Groups 2 through 12, more preferably,Groups 3-12. Even more preferably, the metal is iron, zinc, copper,magnesium, nickel, cobalt, manganese, molybdenum, or selenium, mostpreferably, iron.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium, or the use ofbacteriological media formulated to contain low amounts of a metal. Highmetal conditions are generally present when a chelator is not present inthe medium, a metal is added to the medium, or the combination thereof.Examples of metal chelators include natural and synthetic compounds.Examples of natural compounds include plant phenolic compounds, such asflavenoids, Examples of flavenoids include the copper chelators catechinand naringenin, and the iron chelators myricetin and quercetin. Examplesof synthetic copper chelators include, for instance, tetrathiomolybdate,and examples of synthetic zinc chelators include, for instance, N,N,N′,N′-Tetrakis (2-pyridylmethyl)-ethylene diamine. Examples of syntheticiron chelators include 2,2′-dipyridyl (also referred to in the art asα,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as, the catecholates and hydroxamates, andcitrate. Preferably, 2,2′-dipyridyl is used for the chelation of iron.Typically, 2,2′-dipyridyl is added to the media at a concentration of atleast 0.0025 micrograms/milliliter (μg/ml), at least 0.025 μg/ml, or atleast 0.25 μg/ml, and generally no greater than 10 μg/ml, no greaterthan 20 μg/ml, or no greater than 30 μg/ml.

It is expected that a Yersinia spp. with a mutation in a fur gene willresult in the constitutive expression of many, if not all, of the ironregulated polypeptides of the present invention. The production of a furmutation in a Yersinia spp. can be produced using routine methodsincluding, for instance, transposon, chemical, or site-directedmutagenesis useful for generating gene knock-out mutations in gramnegative bacteria.

The medium used to incubate the microbe and the volume of media used toincubate the microbe can vary. When a microbe is being evaluated for theability to produce one or more of the polypeptides described herein, themicrobe can be grown in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermentor to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermentor areroutine and known in the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, a pH of between 6.5 and 7.5, preferablybetween 6.9 and 7.1, and a temperature of 37° C.

In some aspects of the invention, a microbe may be harvested aftergrowth. Harvesting includes concentrating the microbe into a smallervolume and suspending in a media different than the growth media.Methods for concentrating a microbe are routine and known in the art,and include, for example, filtration or centrifugation. Typically, theconcentrated microbe is suspended in decreasing amounts of buffer.Preferably, the final buffer includes a cation chelator, preferably,ethylenediaminetetraacetic acid (EDTA). An example of a buffer that canbe used contains Tris-base (7.3 grams/liter) and EDTA (0.9 grams/liter),at a pH of 8.5. Optionally, the final buffer also minimizes proteolyticdegradation. This can be accomplished by having the final buffer at a pHof greater than 8.0, preferably, at least 8.5, and/or including one ormore proteinase inhibitors (e.g., phenylmethanesulfonyl fluoride).Optionally and preferably, the concentrated microbe is frozen at −20° C.or below until disrupted.

When the microbe is to be used as a whole cell preparation, theharvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a microbe is to be used toprepare polypeptides of the present invention, the microbe may bedisrupted using chemical, physical, or mechanical methods routine andknown in the art, including, for example, french press, sonication, orhomogenization. Preferably, homogenization is used. An example of asuitable device useful for homogenization is a model C500 AvestinHomogenizer, (Avestin Inc, Ottawa Canada). As used herein, “disruption”refers to the breaking up of the cell. Disruption of a microbe can bemeasured by methods that are routine and known in the art, including,for instance, changes in optical density. Typically, a microbe issubjected to disruption until the percent transmittance is increased by20% when a 1:100 dilution is measured. The temperature during disruptionis typically kept low, preferably at 4° C., to further minimizeproteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, ananionic, zwitterionic, nonionic, or cationic detergent. Preferably, thedetergent is sarcosine, more preferably, sodium lauroyl sarcosinate. Asused herein, the term “solubilize” refers to dissolving cellularmaterials (e.g., polypeptides, nucleic acids, carbohydrates) into theaqueous phase of the buffer in which the microbe was disrupted, and theformation of aggregates of insoluble cellular materials. The conditionsfor solubilization preferably result in the aggregation of polypeptidesof the present invention into insoluble aggregates that are large enoughto allow easy isolation by, for instance, centrifugation.

Significant decreases in LPS are typically observed when the disruptedmicrobe is solubilized in higher levels of sarcosine, solubilized forlonger periods, or the combination thereof. Preferably, the sarcosine isadded such that the final ratio of sarcosine to gram weight of disruptedmicrobe is between 1.0 gram sarcosine per 4.5 grams pellet mass and 6.0grams sarcosine per 4.5 grams pellet mass, preferably, 4.5 gramsarcosine per 4.5 grams pellet mass. The solubilization of the microbemay be measured by methods that are routine and known in the art,including, for instance, changes in optical density. Typically, thesolubilization is allowed to occur for at least 24 hours, preferably, atleast 48 hours, more preferably, at least 72 hours, most preferably, atleast 96 hours. The temperature during disruption is typically kept low,preferably at 4° C.

The insoluble aggregates that include one or more of the polypeptides ofthe present invention may be isolated by methods that are routine andknown in the art. Preferably, the insoluble aggregates are isolated bycentrifugation. Typically, centrifugation of polypeptides that areinsoluble in detergents requires centrifugal forces of at least50,000×g, typically 100,000×g. The use of such centrifugal forcesrequires the use of ultracentrifuges, and scale-up to process largevolumes of sample is often difficult and not economical with these typesof centrifuges. The methods described herein provide for the productionof insoluble aggregates large enough to allow the use of significantlylower centrifugal forces (for instance, 46,000×g). Methods forprocessing large volumes at these lower centrifugal forces are availableand known in the art. Thus, the insoluble aggregates can be isolated ata significantly lower cost. Examples of suitable devices useful forcentrifugation of large volumes include T-1 Sharpies, (Alfa LavalSeparations, Warminster, Pa.) and Hitachi Himac CC40 high speedcentrifuges (Hitachi-Koki Co, Tokyo. Japan).

Optionally and preferably, the sarcosine is removed from the isolatedpolypeptides. Methods for removing sarcosine from the isolatedpolypeptides are known in the art, and include, for instance,diafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, or affinity chromatography, and ultra filtration andwashing the polypeptides in alcohol by diafiltration. After isolation,the polypeptides suspended in buffer and stored at low temperature, forinstance, −20° C. or below.

Polypeptides of the present invention may also be obtained from membersof the genus Yersinia using methods that are known in the art. Theisolation of the polypeptides may be accomplished as described in, forinstance, Emery et al., (U.S. Pat. No. 5,830,479) and Emery et al.,(U.S. Patent Application US 20030036639 A1).

In those aspects of the present invention where a whole cell preparationis to be made, methods known in the art can be used. For instance, aftergrowth a microbe can be killed with the addition of an agent such asglutaraldehyde, formalin, or formaldehyde, at a concentration sufficientto inactivate the cells in the culture. For instance, formalin can beadded at a concentration of 3% (vol:vol). After a period of timesufficient to inactivate the cells, the cells can be harvested by, forinstance, diafiltration and/or centrifugation, and washed.

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. The animal can be, for instance, avian (including,for instance, chickens or turkeys), bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), a companion animal (including, forinstance, cats, dogs, and horses), members of the family Cervidae(including, for instance, deer, elk, moose, caribou, and reindeer),piscine (including, for instance, salmon or trout), crustacean(including, for instance, lobster, crab, or shrimp), members of thefamily Muridae (including, for instance, rats or mice), or human.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at a time after the firstadministration, for instance, 1 to 8 weeks, preferably 2 to 4 weeks,after the first administration of the composition. Subsequent boosterscan be administered one, two, three, four, or more times annually.Without intending to be limited by theory, it is expected that in someaspects of the present invention annual boosters will not be necessary,as an animal will be challenged in the field by exposure to microbesexpressing polypeptides present in the compositions having epitopes thatare identical to or structurally related to epitopes present onpolypeptides of the composition administered to the animal.

In one aspect, the invention is directed to methods for making antibody,such as inducing the production of antibody in an animal, or byrecombinant techniques. The antibody produced includes antibody thatspecifically binds at least one polypeptide present in the composition.In this aspect of the invention, an “effective amount” is an amounteffective to result in the production of antibody in the animal. Methodsfor determining whether an animal has produced antibodies thatspecifically bind polypeptides present in a composition of the presentinvention can be determined as described herein. The present inventionfurther includes antibody that specifically bind to a polypeptide of thepresent invention, and compositions including such antibodies.

The method may be used to produce antibody that specifically bindspolypeptides expressed by a microbe other than the microbe from whichthe polypeptides of the composition were isolated. As used herein, anantibody that can “specifically bind” a polypeptide is an antibody thatinteracts with the epitope of the antigen that induced the synthesis ofthe antibody, or interacts with a structurally related epitope. At leastsome of the polypeptides present in the compositions of the presentinvention typically include epitopes that are conserved in thepolypeptides of different species of microbes. Accordingly, antibodyproduced using a composition derived from one microbe is expected tobind to polypeptides expressed by other microbes and provide broadspectrum protection against gram negative organisms. Examples of gramnegative microbes to which the antibody may specifically bind areenteropathogens, for instance, members of the family Enterobacteriaceae,preferably, members of the genus Yersinia.

The present invention is also directed to the use of such antibody totarget a microbe expressing a polypeptide of the present invention or apolypeptide having an epitope structurally related to an epitope presenton a polypeptide of the present invention. A compound can be covalentlybound to an antibody, where the compound can be, for instance, a toxin.Likewise, such compounds can be covalently bound to a bacterialsiderophore, such as yersiniabactin, to target the microbe. The chemicalcoupling or conjugation of an antibody of the present invention or aportion thereof (such as an Fab fragment) can be carried out using knownand routine methods.

In one aspect the invention is also directed to treating an infection inan animal caused by a gram negative microbe, preferably by a member ofthe genus Yersinia. As used herein, the term “infection” refers to thepresence of a gram negative microbe, preferably, a member of the genusYersinia, in an animal's body, which may or may not be clinicallyapparent. An animal with an infection by member of the genus Yersiniathat is not clinically apparent is often referred to as an asymptomaticcarrier. The method includes administering an effective amount of thecomposition of the present invention to an animal having an infectioncaused by a member of the genus Yersinia, and determining whether theYersinia spp. causing the infection has decreased. Methods fordetermining whether an infection is caused by a member of the genusYersinia are routine and known in the art.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in animals such assheep, cattle, goats, pigs, dogs, birds, rodents and deer that may becaused by infection by a member of the genus Yersinia. Examples ofconditions caused by Yersinia spp. infections include, for instance,diarrhea or enteritis in bovine, ovine, and porcine animals and humans,plague-like illnesses in domestic cats and humans, abortion in cattleand sheep, epididymitis-orchitis in rams, and multiple abscess formationin sheep. Yet another aspect of the present invention is directed attreating cold water diseases of fish such as enteric red mouth diseasein juvenile fish, particularly in intensive aquaculture of trout andsalmon. Treatment of symptoms associated with these conditions can beprophylactic or, alternatively, can be initiated after the developmentof a condition described herein. As used herein, the term “symptom”refers to objective evidence in a subject of a condition caused byinfection by a member of the genus Yersinia spp. Symptoms associatedwith conditions referred to herein and the evaluation of such symptomsare routine and known in the art. Treatment that is prophylactic, forinstance, initiated before a subject manifests symptoms of a conditioncaused by a microbe, is referred to herein as treatment of a subjectthat is “at risk” of developing the condition. Typically, an animal “atrisk” of developing a condition is an animal present in an area wherethe condition has been diagnosed and/or is likely to be exposed to aYersinia spp. causing the condition. Accordingly, administration of acomposition can be performed before, during, or after the occurrence ofthe conditions described herein. Treatment initiated after thedevelopment of a condition may result in decreasing the severity of thesymptoms of one of the conditions, or completely removing the symptoms.In this aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a disease,decrease the severity of the symptoms of a disease, and/or completelyremove the symptoms.

The present invention is also directed to decreasing the colonization bygram negative bacteria, for instance blocking the attachment sites bygram negative bacteria, to tissues of the skeletal system (for instance,bones, cartilage, tendons and ligaments), muscular system, (forinstance, skeletal and smooth muscles), circulatory system (forinstance, heart, blood vessels, capillaries and blood), nervous system(for instance, brain, spinal cord, and peripheral nerves), respiratorysystem (for instance, nose, trachea lungs, bronchi, bronchioceles,alveoli), digestive system (for instance, mouth, salivary glandsoesophagus liver stomach large and small intestine), excretory system(for instance, kidneys, ureters, bladder and urethra), endocrine system(for instance, hypothalamus, pituitary, thyroid, pancreas and adrenalglands), reproductive system (for instance, ovaries, oviduct, uterus,vagina, mammary glands, testes, and seminal vesicles), lymphatic/immunesystems (for instance, lymph, lymph nodes and vessels, mononuclear orwhite blood cells, such as macrophages, neutrophils, monocytes,cosinophils, basophils, lymphocytes t- and b-cells), and specific celllineages (for instance, precursor cells, epithelial cells, stem cells),and the like. Preferably, the gram negative bacteria is a member of thegenus Yersinia. The method includes administering an effective amount ofa composition of the present invention to an animal colonized by, or atrisk of being colonized by a member of the genus Yersinia. In thisaspect of the invention, an “effective amount” is an amount effective todecrease colonization of the animal by the microbe. Methods forevaluating the colonization of an animal by a microbe are routine andknown in the art. For instance, colonization of an animal's intestinaltract by a microbe can be determined by measuring the presence of themicrobe in the animal's feces. It is expected that decreasing thecolonization of an animal by a microbe will reduce transmission of themicrobe to humans.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare antibody compositions from serum, plasma, blood,colostrum, etc. for passive immunization therapies. Antibodycompositions comprising monoclonal antibodies and/or anti-idiotypes canalso be prepared using known methods. Such antibody compositions includechimeric antibodies and humanized antibodies. Chimeric antibodiesinclude human-derived constant regions of both heavy and light chainsand murine-derived variable regions that are antigen-specific (Morrisonet al., Proc. Natl. Acad. Sci. USA, 1984, 81(21):6851-5; LoBuglio etal., Proc. Natl. Acad. Sci. USA, 1989, 86(11):4220-4; Boulianne et al.,Nature, 1984, 312(5995):643-6.). Humanized antibodies substitute themurine constant and framework (FR) (of the variable region) with thehuman counterparts (Jones et al., Nature, 1986, 321(6069):522-5;Riechmann et al., Nature, 1988, 332(6162):323-7; Verhoeyen et al.,Science, 1988, 239(4847): 1534-6; Queen et al., Proc. Natl. Acad. Sci.USA, 1989, 86(24): 10029-33; Daugherty et al., Nucleic Acids Res., 1991,19(9): 2471-6.). Alternatively, certain mouse strains can be used thathave been genetically engineered to produce antibodies that are almostcompletely of human origin; following immunization the B cells of thesemice are harvested and immortalized for the production of humanmonoclonal antibodies (Bruggeman and Taussig, Curr. Opin. Biotechnol.,1997, 8(4):455-8; Lonberg and Huszar. Int. Rev. Immunol., 1995;13(1):65-93; Lonberg et al., Nature, 1994, 368:856-9; Taylor et al.,Nucleic Acids Res., 1992, 20:6287-95.). Passive antibody compositionsand fragments thereof, e.g., scFv, Fab, F(ab)₂ or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods for later use in a concentrated or reconstituted form suchas, for instance, lavage solutions, impregnated dressings and/or topicalagents and the like. Passive immunizing preparations may be particularlyadvantageous for treatment of acute systemic illness, or passiveimmunization of young animals that failed to receive adequate levels ofpassive immunity through maternal colostrum. Antibodies useful forpassive immunization may also be useful to conjugate to various drugs orantibiotics that could be directly targeted to bacteria expressingduring a systemic or localized infection a polypeptide of the presentinvention or a polypeptide having an epitope structurally related to anepitope present on a polypeptide of the present invention.

Animal models, in particular mouse models, are available forexperimentally evaluating the compositions of the present invention(see, for instance, Alpar, H. O., et al., Adv. Drug Deliv. Rev., 51,173-201, (2001), Brem, D., et al., Microbiology, 147, 1115-1127, (2001),Carter, P. B. and F. M. Collins, Infect. Immun., 9, 851-857, (1974),Collyn, F., et al., Infect. Immun., 72.4784-9470, (2004), Di Genaro, M.S., et al., Microbiol. Inununol., 42, 781-788, (1998), Grosfeld, H., etal., Infect Immun, 71, 374-383, (2003), Jones, S. M., et al., Vaccine,19, 358-366, (2001), Karlyshev, A. V., et al., Infect Immun, 69,7810-7819, (2001), Leary, S. E., et al., Microb Pathog, 23, 167-179,(1997), Noll, A., et al., Eur J Immunol, 29, 986-996, (1999), Pelludat,C., et al., Infect Immun, 70, 1832-1841, (2002), Sabhnani. L, et al.,FEMS Immunol Med Microbiol, 38, 215-29, (2003), and Williamson, E. D.,et al., Vaccine, 19, 566-571, (2000)). These mouse models are commonlyaccepted models for the study of human disease caused by members of thegenus Yersinia, and additionally have served as accepted models in thedevelopment and initial testing of vaccines aimed at preventing humanillnesses by Yersinia spp.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically bind polypeptides of the presentinvention, and diagnosing whether an animal may have a condition causedby a microbe expressing polypeptides described herein, or expressingpolypeptides that share epitopes with the polypeptides described herein.Such diagnostic systems may be in kit form. The methods includecontacting an antibody with a preparation that includes polypeptides ofthe present invention to result in a mixture. The antibody may bepresent in a biological sample, for instance, blood, milk, or colostrum.The method further includes incubating the mixture under conditions toallow the antibody to specifically bind the polypeptide to form apolypeptide:antibody complex. As used herein, the term“polypeptide:antibody complex” refers to the complex that results whenan antibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides of the present invention may also includereagents, for instance a buffer, that provide conditions appropriate forthe formation of the polypeptide:antibody complex. Thepolypeptide:antibody complex is then detected. The detection ofantibodies is known in the art and can include, for instance,immunofluorescence and peroxidase. The methods for detecting thepresence of antibodies that specifically bind to polypeptides of thepresent invention can be used in various formats that have been used todetect antibody, including radioimmunoassay and enzyme-linkedimmunosorbent assay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The antibodydetected may be obtained from an animal suspected to have an infectioncaused by a gram negative microbe, more preferably, a member of thefamily Enterobacteriaceae preferably, a member of the genus Yersinia,such as Y. enterocolitica, Y. pseudotuberculosis, or Y. pestis. The kitincludes at least one of the polypeptides of the present invention, or anumber of polypeptides that is an integer greater than 1 (e.g., at least2, at least 3, etc.), in a suitable packaging material in an amountsufficient for at least one assay. Optionally, other reagents such asbuffers and solutions needed to practice the invention are alsoincluded. For instance, a kit may also include a reagent to permitdetection of an antibody that specifically binds to a polypeptide of thepresent invention, such as a detectably labeled secondary antibodydesigned to specifically bind to an antibody obtained from an animal.Instructions for use of the packaged polypeptides are also typicallyincluded. As used herein, the phrase “packaging material” refers to oneor more physical structures used to house the contents of the kit. Thepackaging material is constructed by well known methods, generally toprovide a sterile, contaminant-free environment. The packaging materialmay have a label which indicates that the polypeptides can be used fordetecting antibody that specifically binds polypeptides of the presentinvention. In addition, the packaging material contains instructionsindicating how the materials within the kit are employed to detect theantibody. As used herein, the term “package” refers to a container suchas glass, plastic, paper, foil, and the like, capable of holding withinfixed limits the polypeptides, and other reagents, for instance asecondary antibody. Thus, for example, a package can be a microtiterplate well to which microgram quantities of polypeptides have beenaffixed. A package can also contain a secondary antibody. “Instructionsfor use” typically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Production and Isolation of Metal Regulated Proteins

The compositions used in the following examples were prepared using theproteins derived from Y. enterocolitica ATCC strain 27729 and Y. pestisstrain KIM6+(obtained from R. D. Perry, University of Kentucky). The twostrains were each inoculated from frozen stocks into 25 ml tryptic soybroth (TSB) containing 160 μM 2,2-diprydyl or 300 μM FeCl₃, andincubated at 37° C. while shaking at 400 rpm. Following 12 hours ofincubation, 5 ml of each culture was transferred into 500 ml ofpre-incubated (37° C.) media containing 160 μM 2,2-diprydyl or 300 μMFeCl₃ and incubated at 37° C. while stirring at 100 rpm. After 8 hoursof incubation, the cultures were centrifuged at 10,000×g for 20 minutes.The bacterial pellets were resuspended in 100 ml of sterilephysiological saline and centrifuged at 10,000×g for 10 minutes toremove any contaminating media proteins. The bacterial pellets were thenresuspended in 40 ml of Tris-buffered saline pH 7.2 (TBS) and disruptedby sonication for 1.5 minutes at 4° C. using a Branson 450 equipped witha half inch disruption horn (Branson, Danbury Conn.). The disruptedbacterial suspensions were clarified by centrifugation at 32,000×g for12 minutes. The supernatants were collected and solubilized by theaddition of sodium lauroyl sarcosinate (4% vol/vol) at 4° C. for 24hours. The detergent-insoluble protein-enriched fractions were collectedby centrifugation at 32,000×g for 2.5 hours at 4° C. The protein pelletswere resuspended in 200 μl Tris-buffer (pH 7.2) and stored at −90° C. Asample of each extract was resolved on a 10% SDS-PAGE gel per standardmethods and visualized by Coomassie Blue staining (FIG. 3).

Example 2 Preparation of the Immunizing Compositions Derived from Y.enterocolitica

The proteins made from Y. enterocolitica as described in Example 1 wereused to prepare a composition for administration to animals. Thecomposition contained polypeptides having molecular weights of 268 kDa,92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 45 kDa, 40 kDa, 38 kDa,37 kDa, 31 kDa, or 28 kDa. The polypeptides having molecular weights of83 kDa, 70 kDa, and 66 kDa were expressed only under iron limitedconditions, and the expression of polypeptides having molecular weightsof 268 kDa, 79 kDa, and 45 kDa was enhanced under iron limitedconditions.

A stock vaccine was prepared from the composition by emulsifying theaqueous protein suspension (500 μg total protein/ml) into the commercialadjuvant, EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) using an IKAUltra Turrax T-50 homogenizing vessel (IKA, Cincinnati, Ohio). Thevaccine was administered to mice to give a final dose of 50 μg totalprotein in a 0.1 ml injectable volume with an adjuvant concentration of22.5% vol/vol. A placebo was prepared by replacing the antigen withphysiological saline in the above formulation and emulsifying thesuspension into EMULSIGEN to give an adjuvant concentration of 22.5%.

Example 3 Preparation of Challenge Organism

When used as a challenge, the Y. enterocolitica ATCC strain 27729 wasprepared as follows. Briefly, the isolate from a frozen stock wasstreaked onto a blood agar plate and incubated at 37° C. for 18 hours. Asingle colony was subcultured into 50 ml Tryptic Soy Broth (Difco)containing 25 μg/ml 2, 2′ dipyridyl. The culture was incubated at 37° C.for 6 hours while rotating at 200 rpm, at which point the culture wascentrifuged at 10,000×g for 10 minutes at 4° C. to pellet the bacteria.The bacterial pellet was washed twice by centrifugation in physiologicalsaline at 4° C. The final pellet was resuspended in 25 ml ofphysiological saline and used for challenge. Just prior to challenge, 1ml of the above bacterial suspension was serially diluted ten fold toenumerate the number of CFU/mouse dose.

Example 4 Mouse Vaccination and Challenge Study to Evaluate ProtectionAgainst Intravenous Challenge

The efficacy of the Y. enterocolitica composition was evaluated using alive virulent challenge in mice. Twenty CF-1 mice (Harlan BreedingLaboratories, Indianapolis, Ind.) were divided into two groups of 10mice per group. Mice in the control group were vaccinated with theplacebo, while mice in the second group were immunized with 50 μg of thecomposition obtained as described in Example 1. Immunizations of 0.1 ccwere administered intraperitoneally two times at 14 day intervals.Fourteen days after the second vaccination, a challenge dose of strain27729 (9.4×10 CFU/mouse) was administered to all mice in the lateraltail vein. Mortality was recorded for 7 days following challenge.

Of the 10 placebo-vaccinated mice, 10 (100%) died within 168 hours ofchallenge, while none of the vaccinated mice died within the same timeperiod. Furthermore, all of the vaccinated mice survived for theremainder of the study, which was terminated at 20 days post-challenge.A Kaplan-Meier survival analysis and logrank test (see FIG. 1) indicatedthat immunization provided statistically significant (p<0.0001−)protection against challenge. These results suggest that proteins fromY. enterocolitica grown under iron-restricted conditions constituteeffective antigens in the intravenous mouse model of infection.

Example 5 Western Blot Analysis of Y. enterocolitica Proteins withHyperimmunized and Convalescent Mouse Polyclonal Serum

Western blot analysis was used to evaluate the immuno-reactive proteinsderived from Y. enterocolitica against hyperimmunized mouse polyclonalserum and convalescent sera. Hyperimmunizzed mouse polyclonal serum wasobtained after vaccinating with the composition described in example 2,and convalescent sera was obtained from vaccinated/challenged mice thatsurvived the trial described in example 4. The composition containedpolypeptides having molecular weights of 268 kDa, 92 kDa, 79 kDa, 70kDa, 66 kDa, 54 kDa, 52 kDa, 41 kDa, 38 kDa, 37 kDa, 31 kDa, 28 kDa, andtwo proteins having molecular weights of 83 kDa. The polypeptides havingmolecular weights of 83 kDa, 70 kDa, and 66 kDa were expressed onlyunder iron limited conditions.

To obtain hyper-immunized serum, mice were immunized two times at 14 dayintervals as described in Example 4. The hyperimmunized polyclonal serumwas collected from mice 14 days following the second immunization.Convalescent serum derived from vaccinated/challenged mice was obtained14 days after challenge. The proteins derived from Y. enterocoliticastrain 27729 were first size fractionated on SDS-PAGE (4% stacker/10%resolving gel) using 30 ug total protein as described in example 1. Bandmigration was visualized using broad range kaleidoscope standards(BioRad) to aid in the electroblot transfer while biotinylated broadrange standards were used as molecular weight references on the blot.For western blot analysis, proteins were electroblotted from theSDS-PAGE gel onto trans-blot nitrocellulose membranes (BioRad)overnight, at 4° C. at 50 Volts, in Towbin buffer (25 mM Tris, 192 mMglycine, and 20% methanol) using a BioRad Trans-Blot transfer cell. Thenitrocellulose membrane was blocked by standard methods using 3.0% fishgelatin (BioRad). The hyperimmunized polyclonal serum and convalescentsera was diluted 1/25000 in Tris-buffered saline containing 1.0% fishgelatin, 0.05% tween 20 and 0.2% sodium azide (antibody buffer). Thenitrocellulose membrane was incubated with the primary antibody solutionovernight. The membrane was then washed two times in Tris-BufferedSaline containing 0.05% tween 20 (TTBS) and transferred to antibodybuffer containing a 1/10,000 dilution of goat anti-mouse antibodyconjugated to alkaline phosphatase (BioRad) and a 1/3,000 dilution ofavidin conjugated to alkaline phosphatase (BioRad). The membrane wasincubated at 37° C. for 2 hours on a shaker, and subsequently washed inTTBS four times to remove unbound conjugate. The blot was resolved, for30 minutes at 37° C. on a shaker, in substrate solution containingalkaline phosphate color reagent A and B in 1×AP color developmentbuffer (BioRad).

Western blot analysis was used as a tool to potentially identifyproteins derived from the composition as described in example 1 asimmuno-reactive with antibodies derived from the hyperimmunized and/orconvalescent sera. Western blot analysis revealed a number ofimmuno-reactive proteins. The hyperimmunized sera contained antibodiesthat reacted with proteins at the 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70kDa, 66 kDa, 54 kDa, 52 kDa, 41 kDa, 38 kDa, 37 kDa, 31 kDa and 28 kDa.Similarly, the convalescent sera showed identical banding patterns atthe 268 kDa, 92 kDa, 83 kDa, 79 kDa, 70 kDa, 66 kDa, 54 kDa, 52 kDa, 41kDa, 38 kDa, 37 kDa, 31 kDa and 28 kDa. In addition, threeimmuno-reactive proteins were seen at the 52 kDa, 40 kDa and 20 kDaregions that were not seen on the SDS-PAGE gel initially, nor were theyseen in the western blot analysis using the hyperimmunized sera. It isinteresting to speculate that these three proteins were at too low ofconcentration to be visualized on the SDS-PAGE gel, but may be highlyimmunogenic resulting in greater band intensity after priming the immunesystem that resulted in an enhanced band intensity of these proteinsafter challenge.

The Western Blot analysis of the vaccine composition revealeddifferences in band intensities of the immuno-reactive proteins betweenboth the hyperimmunized and convalescent sera. These differences couldbe the result of different immunogenic properties of individual proteinsand how the immune system recognizes each individual protein within thecomposition. In addition, the amount and ratio of proteins within thecomposition can also influence the immunological status of each proteinwhich can influence the immunological response of the animal toindividual proteins within the composition. Nevertheless, each proteinwithin the composition reacted immunologically as examined by WesternBlot Analysis, thus the immunological response of the mouse uponvaccination, recognized and responded mounting an antibody response toeach individual protein within the composition. Taken together, theresults as described in example 4 illustrate that the proteincomposition was extremely efficacious providing a 100% protection inchallenged mice compared to the non-vaccinated mice having 100%mortality.

Example 6 Western Blot Analysis of Y. pestis Proteins withHyperimmunized Serum Prepared Against Proteins of Y. enterocolitica

Western blot analysis was used to evaluate the immuno-reactive proteinsderived from Y. pestis against hyperimmunized sera prepared against thecomposition derived from Y. enterocolitica as described in example 5.The composition contained polypeptides having molecular weights of 254kDa, 104 kDa, 99 kDa, 94 kDa, 88 kDa, 77 kDa, 73 kDa, 64 kDa, 60 kDa, 46kDa, 44 kDa, 37 kDa 36 kDa, 31 kDa 28 kDa and 20 kDa. The polypeptideshaving molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDawere expressed only under iron limited conditions. The proteins derivedfrom Y. pestis strain KIM6+ was first size fractionated on SDS-PAGE (4%stacker/10% resolving gel) as previously described in example 5 using 30ug total protein. Western blot analysis was run under identicalconditions as described in example 5 except for the followingmodification; the convalescent sera was not tested against the membraneproteins of Y. pestis. The results showed proteins at approximately the254 kDa, 94 kDa, 88 kDa, 46 kDa, 44 kDa, 37 kDa, 36 kDa and 20 kDaregions to be immuno-reactive with antibodies derived from thehyperimmunized serum prepared against membrane proteins of Y.enterocolitica.

Example 7 Mouse Vaccination and Challenge Study to Evaluate ProtectionAgainst Intravenous and Pneumonic Y. pestis Challenge

Eighty-eight female Swiss-Webster (Harlan Laboratories) weighing 16-22grams are equally distributed into 4 groups (22 mice/group), designated1 through 4. The animals are housed in a HEPA-filtered, micro-ventpositive air supply animal caging system (BSL3 facility). Food and waterare supplied ad libitum.

Proteins from Y. pestis strain KIM6+ are prepared as described above inexample 1, and formulated as a vaccine using aluminum hydroxide as theadjuvant (Rehydagel-HPA, Rheis NJ) at a final concentration of 20%vol/vol and 500 μg total protein/ml. The placebo is prepared byreplacing the antigen with PBS while maintaining the same adjuvantconcentration. Mice in Groups 1 and 3 are vaccinated intraperitoneallytwo times at 14 day intervals with 0.1 ml of vaccine containing 50 μgtotal protein, while mice in Groups 2 and 4 are immunized with theplacebo by an identical schedule.

Y. pestis strain CO92 is used for challenge, and is prepared in a BSL3containment facility. Fourteen days after the second vaccination, micein Groups 1 and 2 are challenged intravenously in the lateral tail veinwith 0.1 ml strain CO92 (10³ CFU or approximately 100 LD₅₀ per mouse).Mice in groups 3 and 4 are subjected to an aerosolized challenge dose ofY. pestis CO92 diluted in physiological saline to achieve an approximateconcentration of 100 LD₅₀ CFU per mouse for 30 minutes in an airtightchamber. The aerosolized LD₅₀ for strain CO92 in Swiss Webster mice isdetermined by small pilot studies prior to the proposed challengeexperiments. Mortality is recorded for 21 days after challenge.

Example 8 Fish Vaccination and Challenge Study to Evaluate ProtectionAgainst Y. ruckeri Challenge

Two groups of 20 rainbow trout, designated as groups 1 and 2 weighingapproximately 2 grams are maintained in two separate 60 liter tanks at atemperature of 18° C. Fish are fed twice daily with a commercial troutfeed (Ziegler Brothers, Gardners, Pa.). Fish in group 1 are vaccinatedwith a composition derived from Y. ruckeri using the same method asdescribed in example 1. The extracted proteins derived from Y. ruckeriare used to prepare a vaccine composition for administration to fish. Astock vaccine is prepared from the composition by emulsifying theaqueous protein suspension into a water-in-oil emulsion containingDrakeol 6 mineral oil and Arlacel A as an emulsifier. The vaccine isadministered intraperitoneally to give a final dose of 25 ug totalprotein in a 0.1 cc injectable volume using 0.1 cc. A placebo isprepared by replacing the antigen with physiological saline in the aboveformulation and is given to the fish in group 2 (controls). Fish aregiven a second vaccination 28 days after the first vaccination. Fourteendays after the second vaccination all fish are intraperitoneallychallenged.

A virulent isolate of Y. ruckeri is used for challenge. The challengeisolate is cultured in Tryticase Soy Broth (TSB) containing 160 μM2,2-diprydyl and grown for 12 hours of incubation at 37° C. The cultureis washed once in physiological saline by centrifugation at 10,000×g andresuspended in saline. The culture is adjusted to 5.0×10⁷ CFU per ml.Each trout is intraperitoneally inoculated with 0.1 cc of thecorresponding bacteria at a final challenge dose of 5.0×10⁶ CFU.Mortality was recorded daily for 14 days after challenge. All dead fishare removed from the tank and the livers are removed and plated toenumerate the presence of the challenge organism. Efficacy is measuredas a degree of livability comparing vaccinates to non-vaccinatedcontrols.

Example 9 Characterization of Metal Regulated Proteins of Y.enterocolitica ATCC Strain 27729 and Y. pestis Strain KIM6+

The proteins of the composition prepared as described in example 1 fromY. enterocolitica ATCC strain 27729 and Y. pestis strain KIM6+ werecharacterized using matrix assisted laser desorption/ionizationtime-of-flight spectrometry (MALDI-TOF MS). Samples of each compositionwere was resolved using a 10% sodium dodecyl sulfate-polyacrylamide gel.After the proteins of a composition had been resolved, the gel wasstained with coomasic brilliant blue to visualize the proteins.

Materials and Methods

Excision and Washing.

The gel was washed for 10 minutes with water twice. Each protein band ofinterest was excised by cutting as close to the protein band as possibleto reduce the amount of gel present in the sample. Each gel slice wascut into Ix mm cubes and placed in 1.5 ml tube. The gel pieces werewashed with water for 15 minutes. All the solvent volumes used in thewash steps were approximately equal to twice the volume of the gelslice. The gel slice was next washed with water/acetonitrile (1:1) for15 minutes. The water/acetonitrile mixture was removed, and acetonitrilewas added to cover until the gel pieces turned a sticky white, at whichtime the acetonitrile was removed. The gel pieces were rehydrated in 100mM NH₄HCO₃, and after 5 minutes, a volume of acetonitrile equal to twicethe volume of the gel pieces was added. This was incubated for 15minutes, the liquid removed, and the gel pieces dried in a Speed Vac.

Reduction & Alkylation.

The dried gel pieces were rehydrated in 10 mM DTT and 100 mM NH₄HCO₃,and incubated for 45 minutes at 56° C. After allowing the tubes to coolto room temperature, the liquid was removed and the same volume of amixture of 55 mM iodoacetamide and 100 mM NH₄HCO₃ was immediately added.This was incubated for 30 minutes at room temperature in the dark. Theliquid was removed, acetonitrile was added to cover until the gel piecesturned a sticky white, at which time the acetonitrile was removed. Thegel pieces were rehydrated in 100 mM NH₄HCO₃, and after 5 minutes, avolume of acetonitrile equal to twice the volume of the gel pieces wasadded. This was incubated for 15 minutes, the liquid removed, and thegel pieces dried in a Speed vac. If residual coomassie still remained,the wash with 100 mM NH₄HCO₃/acetonitrile was repeated.

In-Gel Digestion.

Gel pieces were completely dried down in a Speed Vac. The pieces wererehydrated in digestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂, 12.5nanograms per microliter (ng/μl) trypsin) at 4° C. Enough buffer wasadded to cover the gel pieces, and more was added as needed. The gelpieces were incubated on ice for 45 minutes, and the supernatant removedand replaced with 5-2 μl of same buffer without trypsin. This wasincubated at 37° C. overnight in an air incubator.

Extraction of Peptides.

A sufficient volume of 25 mM NH₄HCO₃ was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (in a bathsonicator if possible), and the supernatant was recovered. Theextraction was repeated twice, using 5% formic acid instead of NH₄HCO₃.A sufficient volume of 5% formic acid was added to cover gel pieces, andincubated for 15 minutes (typically in a bath sonicator). The samevolume of acetonitrile was added and incubated for 15 minutes (typicallyin a bath sonicator), and the supernatant was recovered. The extractswere pooled, and 10 mM DTT was added to a final concentration of 1 mMDTT. The sample was dried in a SpeedVac to a final volume ofapproximately 5 μl.

Desalting of Peptides.

The samples were desalted using ZIPTIP pipette tips (C18, Millipore,Billerica, Mass.) as suggested by the manufacturer. Briefly, a samplewas reconstituted in reconstitution solution (5:95 acetonitrile:H₂O,0.1%-0.5% trifluoroacetic acid), centrifuged, and the pH checked toverify that it was less than 3. A ZIPTIP was hydrated by aspirating 10μl of solution 1 (50:50 acetonitrile:H₂O, 0.1% trifluoroacetic acid) anddiscarding the aspirated aliquots. This was followed by aspirating 10 μlof solution 2 (0.1% trifluoroacetic acid in deionized H₂O) anddiscarding the aspirated aliquots. The sample was loaded into the tip byaspirating 10 μl of the sample slowly into the tip, expelling it intothe sample tube, and repeating this 5 to 6 times. Ten microliters ofsolution 2 was aspirated into the tip, the solution discarded byexpelling, and this process was repeated 5-7 times to wash. The peptideswere eluted by aspirating 2.5 μl of ice cold solution 3 (60:40,acetonitrile:H₂O, 0.1% trofluoroacetic acid), expelling, and thenre-aspirating the same aliquot in and out of the tip 3 times. After thesolution has been expelled from the tip, the tube is capped and storedon ice.

Mass Spectrometric Peptide Mapping.

The peptides were suspended in 10 μl to 30 μl of 5% formic acid, andanalyzed by MALDI-TOF MS (Bruker Daltonics Inc., Billerica, Mass.). Themass spectrum of the peptide fragments was determined as suggested bythe manufacturer. Briefly, a sample containing the peptides resultingfrom a tryptic digest were mixed with matrix cyano-4-hydroxycinnamicacid, transferred to a target, and allowed to dry. The dried sample wasplaced in the mass spectrometer, irradiated, and the time of flight ofeach ion detected and used to determine a peptide mass fingerprint foreach protein present in the composition. Known polypeptides (humanangiotensin II, monoisotopic mass MH⁺ 1046.5 (Sigma Chemical Co.), andadenocorticotropin hormone fragment 18-39, MH⁺ 2465.2 (Sigma ChemicalCo.)) were used to standardize the machine.

Data Analysis.

The experimentally observed masses for the peptides in each massspectrum were compared to the expected masses of resulting from knownproteins using the Peptide Mass Fingerprint search method of the Mascotsearch engine (Matrix Science Ltd., London, UK, andwww.matrixscience.com, see Perkins et al., Electrophoresis 20, 3551-3567(1999)). The search parameters included: database, NCBInr; taxonomy,bacteria (eubacteria); type of search, peptide mass fingerprint; enzyme,trypsin; fixed modifications, none; variable modifications, none oroxidized methionine; mass values, monoisotopic; protein mass,unrestricted; peptide mass tolerance, ±1 Da or ±1 330 ppm; peptidecharge state, Mr; max missed cleavages, 1; number of queries, 25.

Results

The result of this search was a mass fingerprint for protein present inthe composition (Tables 5 and 6).

TABLE 5 Experimental data from MALDI-TOF MS analysis of proteinsisolated from Y. enterocolitica ATCC strain 27729. Approximate molecularm/z value of polypeptide Polypeptide weight in kilodaltons fragmentsresulting from Designation (kDa)¹ trypsin digestion² Lw545 268 929.461140.47 1312.57 1440.69 1526.68 1555.66 1581.70 1596.67 1683.69 2110.21Lw391A (±1 Da) 83 687.5 976.4 1001.6 1016.5 1141.6 1170.7 1171.7 1198.51344.5 1357.7 1395.6 1453.7 1477.7 1521.7 1693.8 1716.8 1829.8 1962.02014.1 2020.0 2042.0 2164.1 2226.1 2417.3 3175.5 Lw391B (±1 Da) 831001.6 1104.6 1140.6 1155.5 1171.7 1209.5 1214.7 1338.6 1453.7 1568.81634.9 1651.8 1660.9 1709.8 1750.0 1851.0 1988.1 2105.1 2112.1 2164.12387.2 2453.1 2538.4 3423.7 Lw392 (±1 Da 79 837.5 1018.6 1071.5 1086.51132.7 1189.5 1215.6 1236.6 1256.6 1264.6 1361.6 1497.7 1502.8 1615.71653.8 1718.9 1770.9 1820.9 1828.1 2006.0 2067.1 2120.9 2300.3 2308.22783.3 2912.4 3024.5 3287.6 Lw393 (±1 Da) 70 714.6 760.5 807.5 820.5920.5 1024.6 1052.6 1187.6 1200.6 1395.7 1437.7 1480.7 1541.9 1546.91565.8 1668.8 1732.0 1790.9 1906.0 1982.2 1984.1 1997.1 2011.1 2028.22060.2 2134.1 2163.3 2275.4 2364.3 2378.5 2384.3 2564.4 2658.4 2834.72930.7 Lw550 66 868.6500 882.5700 884.5900 1021.7000 1087.7100 1168.73001177.8200 1208.6800 1346.8700 1750.0100 1755.0500 1852.2800 2521.81002607.6700 2944.1000 3087.0800 Lw552 45 1140.5500 1209.5400 1312.58001440.6400 1501.6900 1526.6200 1581.6800 1596.6800 Lw555 37 705.3700881.2400 971.1700 1122.3100 1280.1900 1295.2200 1335.2900 1510.30001908.5300 2245.7300 2324.7100 2642.7500 2985.0200 3087.9700 Lw557 31864.49 1404.50 1616.68 1780.68 1876.82 2071.04 2379.08 ¹Molecularweight, in kilodaltons, of polypeptide obtained from Y. enterocoliticaATCC strain 27729. ²m/z, mass (m) to charge (z) ratio.

TABLE 6 Experimental data from MALDI-TOF MS analysis of proteinsisolated from Y. pestis strain KIM6+. Approximate molecular m/z value ofpolypeptide Polypeptide weight in kilodaltons fragments resulting fromDesignation (kDa)¹ trypsin digestion² Lw529 104 644.50 685.40 771.40841.40 899.50 962.40 1137.40 1277.40 1293.40 1386.40 1410.50 1422.601498.60 1567.50 1679.70 1684.60 1726.70 1873.70 1991.70 2020.80 2182.802584.90 2843.20 Lw530 99 1191.40 1514.50 1591.50 1597.50 1637.50 1671.501714.60 1719.60 1751.60 1820.60 1863.70 1967.60 2122.60 Lw531 94 962.201168.20 1258.30 1372.30 1384.30 1409.30 1521.40 1669.50 1686.40 1714.401717.40 1797.50 1833.50 1845.50 2218.60 2426.60 Lw532 88 889.30 927.30946.40 961.40 1172.40 1177.40 1290.40 1333.50 1358.40 1404.50 1419.501508.50 1579.60 1673.60 1736.60 2401.00 2666.00 Lw533 77 687.40 785.40859.30 953.40 1141.50 1156.50 1171.50 1198.40 1403.50 1409.50 1483.501523.50 1551.60 1618.60 1675.50 1746.60 1788.70 1820.70 1852.80 1941.602013.90 2018.80 2057.80 2168.80 2170.00 2427.00 2457.80 2829.10 Lw534 73629.40 749.40 910.30 931.40 1292.50 1371.50 1441.40 1479.50 1587.601605.60 1641.60 1655.50 1706.60 1708.60 1758.70 1797.80 1856.80 1913.702004.80 2072.80 2155.90 2301.90 2395.90 2484.90 2558.20 2676.20 2984.103162.30 3185.30 3425.50 3472.40 Lw535 64 714.40 760.40 774.40 807.40920.40 1024.40 1052.40 1103.40 1165.40 1187.40 1200.40 1282.50 1395.401445.50 1480.50 1546.60 1668.50 1693.60 1731.60 1790.60 1905.70 1969.701981.80 2010.80 2027.80 2059.80 2163.00 2363.90 2378.10 2820.20 2930.20Lw536 60 1011.46 1187.55 1231.54 1238.57 1291.57 1567.76 1605.78 1621.741669.68 2021.02 2397.21 Lw537 46 873.53 1001.53 1180.50 1258.60 1300.671307.58 1325.59 1368.72 1395.70 1436.67 1609.91 1616.82 1780.94 1952.051959.02 2020.04 2099.15 2178.22 2710.51 Lw538 44 776.51 837.65 905.621027.71 1073.74 1200.79 1232.67 1233.72 1290.81 1376.71 1603.90 1615.011711.08 1774.04 1796.13 1906.14 1978.16 2001.23 Lw683 37 691.26 894.21911.21 1050.26 1115.19 1120.19 1122.24 1198.17 1263.19 1308.24 1320.341423.28 1437.31 1491.23 1534.41 1579.39 2245.71 2367.68 2487.63 2684.792980.02 3292.91 Lw541 31 1020.84 1075.77 1203.86 1248.88 1322.87 1404.951788.29 1991.60 2092.61 2119.74 Lw542 31 1143.91 1299.97 1309.09 1341.971372.04 1580.12 1781.45 1791.43 1954.57 2191.78 2632.11 Lw544 20 807.401114.43 1210.48 1244.46 1259.51 1270.49 1357.49 1790.90 2003.91 2989.45¹Molecular weight, in kilodaltons, of polypeptide obtained from Y.pestis strain KIM6+. ²m/z, mass (m) to charge (z) ratio.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A composition comprising: a Yersinia spp. microbe comprising polypeptides expressed by the Yersinia spp. when incubated in media comprising an iron chelator and not expressed at detectable levels when the Yersinia spp. is grown in the media without the iron chelator; and an adjuvant.
 2. The composition of claim 1 wherein the Yersinia spp. comprises Y. pestis.
 3. The composition of claim 2 wherein the Y. pestis comprises Y. pestis strain KIM6+.
 4. The composition of claim 2 wherein the polypeptides comprise: a first polypeptide having a molecular weight of 94 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; a second polypeptide having a molecular weight of 88 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; a third polypeptide having a molecular weight of 77 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; a fourth polypeptide having a molecular weight of 73 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; and a fifth polypeptide having a molecular weight of 64 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel.
 5. The composition of claim 4 wherein: the 94 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:241, and SEQ ID NO:242; the 88 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, and SEQ ID NO:259; the 77 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, and SEQ ID NO:284; the 73 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:202, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, and SEQ ID NO:318; and the 64 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:334, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO:344, SEQ ID NO:345, SEQ ID NO:348, and SEQ ID NO:349.
 6. A composition comprising: isolated polypeptides having molecular weights of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa, wherein molecular weight is determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel, wherein the polypeptides having a molecular weight of 94 kDa, 88 kDa, 77 kDa, 73 kDa, and 64 kDa are expressed by a Yersinia pestis at a greater level when incubated in media comprising an iron chelator than when grown in the media without the iron chelator; wherein: the 94 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:241, and SEQ ID NO:242; the 88 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256, SEQ ID NO:257, SEQ ID NO:258, and SEQ ID NO:259; the 77 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:261, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:265, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271, SEQ ID NO:272, SEQ ID NO:273, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:279, SEQ ID NO:280, SEQ ID NO:281, SEQ ID NO:282, SEQ ID NO:283, and SEQ ID NO:284; the 73 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:290, SEQ ID NO:291, SEQ ID NO:202, SEQ ID NO:293, SEQ ID NO:294, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:298, SEQ ID NO:299, SEQ ID NO:300, SEQ ID NO:301, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:305, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:309, SEQ ID NO:310, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:314, SEQ ID NO:315, and SEQ ID NO:318; and the 64 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:319, SEQ ID NO:320, SEQ ID NO:321, SEQ ID NO:322, SEQ ID NO:323, SEQ ID NO:324, SEQ ID NO:325, SEQ ID NO:326, SEQ ID NO:327, SEQ ID NO:328, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:334, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:342, SEQ ID NO:343, SEQ ID NO:344, SEQ ID NO:345, SEQ ID NO:348, and SEQ ID NO:349; and an adjuvant.
 7. The composition of claim 2 further comprising a pharmaceutically acceptable carrier.
 8. The composition of claim 1 wherein the Yersinia spp. comprises Y. enterocolitica.
 9. The composition of claim 8 wherein the Yersinia spp. comprises Y. enterocolitica ATCC strain
 27729. 10. The composition of claim 8 wherein the polypeptides comprise: a first polypeptide having a molecular weight of 83 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; a second polypeptide having a molecular weight of 70 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel; and a third polypeptide having a molecular weight of 66 kDa as determined by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel.
 11. The composition of claim 10 wherein: the 83 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58, or the amino acid sequences of SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82 and one selected from the group consisting of: SEQ ID NO:67 and SEQ ID NO:78; the 70 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:144, and SEQ ID NO:145; and the 66 kDa polypeptide comprises the amino acid sequences of SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:150 SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, and SEQ ID NO:161.
 12. The composition of claim 1 wherein the Yersinia spp. microbe is inactivated. 